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Sample records for actin thin filaments

  1. Contractile properties of thin (actin) filament-reconstituted muscle fibers.

    PubMed

    Ishiwata, S; Funatsu, T; Fujita, H

    1998-01-01

    Selective removal and reconstitution of the components of muscle fibers (fibrils) is a useful means of examining the molecular mechanism underlying the formation of the contractile apparatus. In addition, this approach is powerful for examining the structure-function relationship of a specific component of the contractile system. In previous studies, we have achieved the partial structural and functional reconstitution of thin filaments in the skeletal contractile apparatus and full reconstitution in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with plasma gelsolin, an actin filament-severing protein. Under these conditions, no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted by polymerization on the short actin fragments remaining at the Z line, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached as high as 30% of the original level in skeletal muscle, while it reached 140% in cardiac muscle. The augmentation of tension in cardiac muscle is mainly attributable to the elongation of reconstituted filaments, longer than the average length of thin filaments in an intact muscle. These results indicate that a muscle contractile apparatus with a high order structure and function can be constructed by the self-assembly of constituent proteins. Recently, we applied this reconstitution system to the study of the mechanism of spontaneous oscillatory contraction (SPOC) in thin (actin) filament-reconstituted cardiac muscle fibers. As a result, we found that SPOC occurs even in regulatory protein-free actin filament-reconstituted fibers (Fujita & Ishiwata, manuscript submitted), although the SPOC conditions were slightly different from the standard SPOC conditions. This result strongly suggests that spontaneous oscillation

  2. Gestalt-binding of tropomyosin on actin during thin filament activation.

    PubMed

    Lehman, William; Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan; Raunser, Stefan

    2013-08-01

    Our thesis is that thin filament function can only be fully understood and muscle regulation then elucidated if atomic structures of the thin filament are available to reveal the positions of tropomyosin on actin in all physiological states. After all, it is tropomyosin influenced by troponin that regulates myosin-crossbridge cycling on actin and therefore controls contraction in all muscles. In addition, we maintain that a complete appreciation of thin filament activation also requires that the mechanical properties of tropomyosin itself are recognized and then related to the effect of myosin-association on actin. Taking the Gestalt-binding of tropomyosin into account, coupled with our electron microscopy structures and computational chemistry, we propose a comprehensive mechanism for tropomyosin regulatory movement over the actin filament surface that explains the cooperative muscle activation process. In fact, well-known point mutations of critical amino acids on the actin-tropomyosin binding interface disrupt Gestalt-binding and are associated with a number of inherited myopathies. Moreover, dysregulation of tropomyosin may also be a factor that interferes with the gatekeeping operation of non-muscle tropomyosin in the controlling interactions of a wide variety of cellular actin-binding proteins. The clinical relevance of Gestalt-binding is discussed in articles by the Marston and the Gunning groups in this special journal issue devoted to the impact of tropomyosin on biological systems.

  3. Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments.

    PubMed

    Tao, T; Gong, B J; Leavis, P C

    1990-03-16

    The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between the cysteine residue at position 133 (Cys133) of troponin-I and Cys374 of actin increases by approximately 15 angstroms on binding of Ca2+ to troponin-C. Also, troponin-I labeled at Cys133 with benzophenone-4-maleimide could be photo cross-linked to actin in the absence of Ca2+, but not in its presence. These results suggest that troponin-I is attached to actin in the Ca2(+)-free or relaxed state of muscle, and that it detaches from actin on Ca2+ activation of contraction. Thus, troponin-I may function as a Ca2(+)-dependent molecular switch in regulation of skeletal muscle contraction.

  4. Impaired tropomyosin-troponin interactions reduce activation of the actin thin filament.

    PubMed

    Robaszkiewicz, Katarzyna; Ostrowska, Zofia; Cyranka-Czaja, Anna; Moraczewska, Joanna

    2015-05-01

    Tropomyosin and troponin are bound to the actin filament to control the contraction of striated muscle in the Ca-dependent manner. The interactions between both regulatory proteins important for the regulation process are not fully understood. To gain more insight into the mechanisms of the thin filament regulation by skeletal α-tropomyosin and troponin, we analyzed effects of seven myopathy-related substitutions: Leu99Met, Ala155Thr, Arg167Gly, Arg167Cys, Arg167His, Lys168Glu, and Arg244Gly. All substitutions reduced Ca-dependent activation of the actomyosin ATPase. The effects of mutations in Arg167 and Lys168 were the most severe. The amino acid substitutions did not significantly affect troponin binding to the whole filament, but reduced 1.2-2.8 fold the affinity of troponin to tropomyosin alone. The excimer fluorescence of N-(1-pyrene)iodoacetamide, a probe attached to the central Cys190, demonstrated that substitutions located near the troponin core domain-binding region strongly affected conformational changes accompanying the tropomyosin-troponin interactions. The thermal stability of all tropomyosin mutants was lower than the stability of the wild type tropomyosin, with TM reduced by 5.3-8.5°C. Together the analyses demonstrated that the myopathy-causing mutations affected tropomyosin structure and led to changes in interactions between tropomyosin and troponin, which impaired the transition of the thin filament from the inactive off to the active on state.

  5. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  6. Association of actin filaments with axonal microtubule tracts.

    PubMed

    Bearer, E L; Reese, T S

    1999-02-01

    Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 microm with some at least as long as 3.5 microm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.

  7. Actin filament curvature biases branching direction

    NASA Astrophysics Data System (ADS)

    Wang, Evan; Risca, Viviana; Chaudhuri, Ovijit; Chia, Jia-Jun; Geissler, Phillip; Fletcher, Daniel

    2012-02-01

    Actin filaments are key components of the cellular machinery, vital for a wide range of processes ranging from cell motility to endocytosis. Actin filaments can branch, and essential in this process is a protein complex known as the Arp2/3 complex, which nucleate new ``daughter'' filaments from pre-existing ``mother'' filaments by attaching itself to the mother filament. Though much progress has been made in understanding the Arp2/3-actin junction, some very interesting questions remain. In particular, F-actin is a dynamic polymer that undergoes a wide range of fluctuations. Prior studies of the Arp2/3-actin junction provides a very static notion of Arp2/3 binding. The question we ask is how differently does the Arp2/3 complex interact with a straight filament compared to a bent filament? In this study, we used Monte Carlo simulations of a surface-tethered worm-like chain to explore possible mechanisms underlying the experimental observation that there exists preferential branch formation by the Arp2/3 complex on the convex face of a curved filament. We show that a fluctuation gating model in which Arp2/3 binding to the actin filament is dependent upon a rare high-local-curvature shape fluctuation of the filament is consistent with the experimental data.

  8. Reconstitution of a Minimal Actin Cortex by Coupling Actin Filaments to Reconstituted Membranes.

    PubMed

    Vogel, Sven K

    2016-01-01

    A thin layer of actin filaments in many eukaryotic cell types drives pivotal aspects of cell morphogenesis and is generally cited as the actin cortex. Myosin driven contractility and actin cytoskeleton membrane interactions form the basis of fundamental cellular processes such as cytokinesis, cell migration, and cortical flows. How the interplay between the actin cytoskeleton, the membrane, and actin binding proteins drives these processes is far from being understood. The complexity of the actin cortex in living cells and the hardly feasible manipulation of the omnipotent cellular key players, namely actin, myosin, and the membrane, are challenging in order to gain detailed insights about the underlying mechanisms. Recent progress in developing bottom-up in vitro systems where the actin cytoskeleton is combined with reconstituted membranes may provide a complementary route to reveal general principles underlying actin cortex properties. In this chapter the reconstitution of a minimal actin cortex by coupling actin filaments to a supported membrane is described. This minimal system may be very well suited to study for example protein interactions on membrane bound actin filaments in a very controlled and quantitative manner as it may be difficult to perform in living systems.

  9. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  10. Effects of filament rigidity in myosin II-induced actin network contractility and dynamics

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Gardel, Margaret

    2014-03-01

    Cells change shape, deforming to move and divide. The dynamic protein scaffold that shapes the cell is the cortex, a disordered, thin network of actin filaments. Random, local stresses generated by myosin II in the network create cellular-scale deformations. Myosin induced buckling and severing of actin filaments has been shown to underlie the contractility of two-dimensional disordered actin networks. This non-linear elastic response of actin filaments is thought to be an essential symmetry breaking mechanism to produce robust contractility in disordered actomyosin networks. To test this idea, we explore the effects of an actin bundling protein fascin, a crosslinker which induces polarity specific bundling of actin filaments, to create a network of F-actin bundles. We investigate myosin-induced stresses in a network of randomly oriented actin filaments, confined to a thin sheet at a supported lipid bilayer surface through a crowding agent. We find fascin-bundled filaments are less prone to filament buckling and show increased filament sliding, causing the myosin activity to induce network reorganization rather than contraction. Thus, changes in the filament bending rigidity in motor-filament systems can drive the system between distinct states with unique dynamic and mechanical signatures.

  11. Mechanism of Actin Filament Bundling by Fascin

    SciTech Connect

    Jansen, Silvia; Collins, Agnieszka; Yang, Changsong; Rebowski, Grzegorz; Svitkina, Tatyana; Dominguez, Roberto

    2013-03-07

    Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four {beta}-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in {beta}-trefoil domains 1 and 3. The site in {beta}-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in {beta}-trefoil-3 is related by pseudo-2-fold symmetry to that in {beta}-trefoil-1. The two sites are {approx}5 nm apart, resulting in a distance between actin filaments in the bundle of {approx}8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

  12. A Robust Actin Filaments Image Analysis Framework

    PubMed Central

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts

  13. Formation and Destabilization of Actin Filaments with Tetramethylrhodamine-Modified Actin

    PubMed Central

    Kudryashov, Dmitry S.; Phillips, Martin; Reisler, Emil

    2004-01-01

    Actin labeling at Cys374 with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics. PMID:15298916

  14. Self-assembly of Artificial Actin Filaments

    NASA Astrophysics Data System (ADS)

    Grosenick, Christopher; Cheng, Shengfeng

    Actin Filaments are long, double-helical biopolymers that make up the cytoskeleton along with microtubules and intermediate filaments. In order to further understand the self-assembly process of these biopolymers, a model to recreate actin filament geometry was developed. A monomer in the shape of a bent rod with vertical and lateral binding sites was designed to assemble into single or double helices. With Molecular Dynamics simulations, a variety of phases were observed to form by varying the strength of the binding sites. Ignoring lateral binding sites, we have found a narrow range of binding strengths that lead to long single helices via various growth pathways. When lateral binding strength is introduced, double helices begin to form. These double helices self-assemble into substantially more stable structures than their single helix counterparts. We have found double helices to form long filaments at about half the vertical binding strength of single helices. Surprisingly, we have found that triple helices occasionally form, indicating the importance of structural regulation in the self-assembly of biopolymers.

  15. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

  16. Actin filaments as dynamic reservoirs for Drp1 recruitment

    PubMed Central

    Hatch, Anna L.; Ji, Wei-Ke; Merrill, Ronald A.; Strack, Stefan; Higgs, Henry N.

    2016-01-01

    Drp1 is a dynamin-family GTPase recruited to mitochondria and peroxisomes, where it oligomerizes and drives membrane fission. Regulation of mitochondrial Drp1 recruitment is not fully understood. We previously showed that Drp1 binds actin filaments directly, and actin polymerization is necessary for mitochondrial Drp1 oligomerization in mammals. Here we show the Drp1/actin interaction displays unusual properties that are influenced by several factors. At saturation, only a fraction Drp1 binds actin filaments, and the off-rate of actin-bound Drp1 is significantly increased by unbound Drp1. GDP and GTP accelerate and decelerate Drp1/actin binding dynamics, respectively. Actin has a biphasic effect on Drp1 GTP hydrolysis, increasing at low actin:Drp1 ratio but returning to baseline at high ratio. Drp1 also bundles filaments. Bundles have reduced dynamics but follow the same trends as single filaments. Drp1 preferentially incorporates into bundles at higher ionic strength. We measure Drp1 concentration to be ∼0.5 μM in U2OS cell cytosol, suggesting the actin-binding affinity measured here (Kd = 0.6 μM) is in the physiologically relevant range. The ability of Drp1 to bind actin filaments in a highly dynamic manner provides potential for actin filaments to serve as reservoirs of oligomerization-competent Drp1 that can be accessed for mitochondrial fission. PMID:27559132

  17. Geometrical and Mechanical Properties Control Actin Filament Organization

    PubMed Central

    Ennomani, Hajer; Théry, Manuel; Nedelec, Francois; Blanchoin, Laurent

    2015-01-01

    The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model. PMID:26016478

  18. Geometrical and mechanical properties control actin filament organization.

    PubMed

    Letort, Gaëlle; Politi, Antonio Z; Ennomani, Hajer; Théry, Manuel; Nedelec, Francois; Blanchoin, Laurent

    2015-05-01

    The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model.

  19. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed

    Fujita, H; Ishiwata, S

    1998-09-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.

  20. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed Central

    Fujita, H; Ishiwata, S

    1998-01-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself. PMID:9726945

  1. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

    PubMed

    Niederman, R; Amrein, P C; Hartwig, J

    1983-05-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

  2. Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments.

    PubMed

    Jiwani, Shahanawaz; Alvarado, Stephenie; Ohr, Ryan J; Romero, Adriana; Nguyen, Brenda; Jewett, Travis J

    2013-02-01

    All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells.

  3. Regulation of Sodium Channel Activity by Capping of Actin Filaments

    PubMed Central

    Shumilina, Ekaterina V.; Negulyaev, Yuri A.; Morachevskaya, Elena A.; Hinssen, Horst; Khaitlina, Sofia Yu

    2003-01-01

    Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells. PMID:12686620

  4. Mechanism of interaction of Dictyostelium severin with actin filaments

    PubMed Central

    1982-01-01

    Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo. PMID:6897549

  5. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  6. Direct Observation of Tropomyosin Binding to Actin Filaments

    PubMed Central

    Schmidt, William M.; Lehman, William; Moore, Jeffrey R.

    2015-01-01

    Tropomyosin is an elongated α-helical coiled-coil that binds to seven consecutive actin subunits along the long-pitch helix of actin filaments. Once bound, tropomyosin polymerizes end-to-end and both stabilizes F-actin and regulates access of various actin binding proteins including myosin in muscle and non-muscle cells. Single tropomyosin molecules bind weakly to F-actin with millimolar Kd, whereas the end-to-end linked tropomyosin associates with about a one thousand-fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In the current study, we used total internal reflection fluorescence (TIRF) microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin-stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites following random low affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on tropomyosin concentration, occurring at approximately 100 monomers/(μM*s). The random tropomyosin binding to F-actin leads to discontinuous end-to-end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin-bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process. PMID:26033920

  7. Formation of actin filament bundles in the ring canals of developing Drosophila follicles

    PubMed Central

    1996-01-01

    Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay

  8. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

    PubMed Central

    1996-01-01

    Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo. PMID:8947553

  9. Evidence for filamentous actin in ookinetes of a malarial parasite.

    PubMed

    Siden-Kiamos, Inga; Louis, Christos; Matuschewski, Kai

    2012-02-01

    Extracellular stages of apicomplexan parasites utilize their own actin myosin motor machinery for gliding locomotion, penetration of cell barriers, and host cell invasion. Thus far, filamentous actin could not be visualized by standard microscopic techniques in vivo. Here, we describe the generation of a novel peptide antibody against the divergent amino-terminal portion of the major Plasmodium isoform, actin I. We show that our antiserum, termed Ab-actinI-I, is conformation-specific. In motile ookinetes it recognizes actin in rod-like structures, which are sensitive to inhibitors interfering with actin polymerization. The average size of the rods is 600 nm, which is considerably longer than what has been detected in in vitro studies of actin filaments.

  10. Actin binding domain of filamin distinguishes posterior from anterior actin filaments in migrating Dictyostelium cells

    PubMed Central

    Shibata, Keitaro; Nagasaki, Akira; Adachi, Hiroyuki; Uyeda, Taro Q. P.

    2016-01-01

    Actin filaments in different parts of a cell interact with specific actin binding proteins (ABPs) and perform different functions in a spatially regulated manner. However, the mechanisms of those spatially-defined interactions have not been fully elucidated. If the structures of actin filaments differ in different parts of a cell, as suggested by previous in vitro structural studies, ABPs may distinguish these structural differences and interact with specific actin filaments in the cell. To test this hypothesis, we followed the translocation of the actin binding domain of filamin (ABDFLN) fused with photoswitchable fluorescent protein (mKikGR) in polarized Dictyostelium cells. When ABDFLN-mKikGR was photoswitched in the middle of a polarized cell, photoswitched ABDFLN-mKikGR rapidly translocated to the rear of the cell, even though actin filaments were abundant in the front. The speed of translocation (>3 μm/s) was much faster than that of the retrograde flow of cortical actin filaments. Rapid translocation of ABDFLN-mKikGR to the rear occurred normally in cells lacking GAPA, the only protein, other than actin, known to bind ABDFLN. We suggest that ABDFLN recognizes a certain feature of actin filaments in the rear of the cell and selectively binds to them, contributing to the posterior localization of filamin.

  11. Mechanical Heterogeneity Favors Fragmentation of Strained Actin Filaments

    PubMed Central

    De La Cruz, Enrique M.; Martiel, Jean-Louis; Blanchoin, Laurent

    2015-01-01

    We present a general model of actin filament deformation and fragmentation in response to compressive forces. The elastic free energy density along filaments is determined by their shape and mechanical properties, which were modeled in terms of bending, twisting, and twist-bend coupling elasticities. The elastic energy stored in filament deformation (i.e., strain) tilts the fragmentation-annealing reaction free-energy profile to favor fragmentation. The energy gradient introduces a local shear force that accelerates filament intersubunit bond rupture. The severing protein, cofilin, renders filaments more compliant in bending and twisting. As a result, filaments that are partially decorated with cofilin are mechanically heterogeneous (i.e., nonuniform) and display asymmetric shape deformations and energy profiles distinct from mechanically homogenous (i.e., uniform), bare actin, or saturated cofilactin filaments. The local buckling strain depends on the relative size of the compliant segment as well as the bending and twisting rigidities of flanking regions. Filaments with a single bare/cofilin-decorated boundary localize energy and force adjacent to the boundary, within the compliant cofilactin segment. Filaments with small cofilin clusters were predicted to fragment within the compliant cofilactin rather than at boundaries. Neglecting contributions from twist-bend coupling elasticity underestimates the energy density and gradients along filaments, and thus the net effects of filament strain to fragmentation. Spatial confinement causes compliant cofilactin segments and filaments to adopt higher deformation modes and store more elastic energy, thereby promoting fragmentation. The theory and simulations presented here establish a quantitative relationship between actin filament fragmentation thermodynamics and elasticity, and reveal how local discontinuities in filament mechanical properties introduced by regulatory proteins can modulate both the severing efficiency

  12. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  13. Actin Filament Stress Fibers in Vascular Endothelial Cells in vivo

    NASA Astrophysics Data System (ADS)

    Wong, Albert J.; Pollard, Thomas D.; Herman, Ira M.

    1983-02-01

    Fluorescence microscopy with 7-nitrobenz-2-oxa-3-diazole phallacidin was used to survey vertebrate tissues for actin filament bundles comparable to the stress fibers of cultured cells. Such bundles were found only in vascular endothelial cells. Like the stress fibers of cultured cells, these actin filament bundles were stained in a punctate pattern by fluorescent antibodies to both alpha-actinin and myosin. The stress fibers were oriented parallel to the direction of blood flow and were prominent in endothelial cells from regions exposed to high-velocity flow, such as the left ventricle, aortic valve, and aorta. Actin bundles may help the endothelial cell to withstand hemodynamic stress.

  14. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

    2001-01-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

  15. Cofilin cooperates with fascin to disassemble filopodial actin filaments

    PubMed Central

    Breitsprecher, Dennis; Koestler, Stefan A.; Chizhov, Igor; Nemethova, Maria; Mueller, Jan; Goode, Bruce L.; Small, J. Victor; Rottner, Klemens; Faix, Jan

    2011-01-01

    Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin. PMID:21940796

  16. F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments

    PubMed Central

    1996-01-01

    The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper. PMID:8947552

  17. Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

    SciTech Connect

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C.; Navaza, Jorge; Dominguez, Roberto

    2013-11-20

    Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin {beta}4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin {beta}4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  18. Structure of a longitudinal actin dimer assembled by tandem w domains: implications for actin filament nucleation.

    PubMed

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C; Navaza, Jorge; Dominguez, Roberto

    2010-10-15

    Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  19. Functional synergy of actin filament cross-linking proteins.

    PubMed

    Tseng, Yiider; Schafer, Benjamin W; Almo, Steven C; Wirtz, Denis

    2002-07-12

    The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.

  20. Correlative nanoscale imaging of actin filaments and their complexes

    NASA Astrophysics Data System (ADS)

    Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil; Gimzewski, James K.

    2013-06-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

  1. Multiple crystal structures of actin dimers and their implications for interactions in the actin filament

    PubMed Central

    Sawaya, Michael R.; Kudryashov, D. S.; Pashkov, Inna; Adisetiyo, Helty; Reisler, Emil; Yeates, Todd O.

    2008-01-01

    The structure of actin in its monomeric form is known at high resolution, while the structure of filamentous F-actin is only understood at considerably lower resolution. Knowing pre­cisely how the monomers of actin fit together would lead to a deeper understanding of the dynamic behavior of the actin filament. Here, a series of crystal structures of actin dimers are reported which were prepared by cross-linking in either the longitudinal or the lateral direction in the filament state. Laterally cross-linked dimers, comprised of monomers belonging to different protofilaments, are found to adopt configurations in crystals that are not related to the native structure of filamentous actin. In contrast, multiple structures of longitudinal dimers consistently reveal the same interface between monomers within a single protofilament. The re­appearance of the same longitudinal interface in multiple crystal structures adds weight to arguments that the interface visualized is similar to that in actin filaments. Highly conserved atomic interactions involving residues 199–205 and 287–291 are highlighted. PMID:18391412

  2. Regulation of actin filament length in erythrocytes and striated muscle.

    PubMed

    Fowler, V M

    1996-02-01

    Actin filaments polymerize in vitro to lengths which display an exponential distribution, yet in many highly differentiated cells they can be precisely maintained at uniform lengths in elaborate supramolecular structures. Recent results obtained using two classic model systems, the erythrocyte membrane cytoskeleton and the striated muscle sarcomere, reveal surprising similarities and instructive differences in the molecules and mechanisms responsible for determining and maintaining actin filament lengths in these two systems. Tropomodulin caps the slow-growing, pointed filament ends in muscle and in erythrocytes. CapZ caps the fast-growing, barbed filament ends in striated muscle, whereas a newly discovered barbed end capping protein, adducin, may cap the barbed filament ends in erythrocytes. The mechanisms responsible for specifying the characteristic filament lengths in these systems are more elusive and may include strict control of the relative amounts of actin filament capping proteins and side-binding proteins, molecular templates (e.g. tropomyosin and nebulin) and/or verniers (e.g. tropomyosin).

  3. Accelerated actin filament polymerization from microtubule plus-ends

    PubMed Central

    Henty-Ridilla, Jessica L.; Rankova, Aneliya; Eskin, Julian A.; Kenny, Katelyn; Goode, Bruce L.

    2016-01-01

    Microtubules govern actin network remodeling in a wide range of biological processes, yet the mechanisms underlying this cytoskeletal crosstalk have remained obscure. Here we used single-molecule fluorescence microscopy to show that the microtubule plus-end associated protein CLIP-170 binds tightly to formins to accelerate actin filament elongation. Furthermore, we observed mDia1 dimers and CLIP-170 dimers co-tracking growing filament ends for minutes. CLIP-170-mDia1 complexes promoted actin polymerization approximately 18 times faster than free barbed end growth, while simultaneously enhancing protection from capping protein. We used a microtubule-actin dynamics co-reconstitution system to observe CLIP-170-mDia1 complexes being recruited to growing microtubule ends by EB1. The complexes triggered rapid growth of actin filaments that remained attached to the microtubule surface. These activities of CLIP-170 were required in primary neurons for normal dendritic morphology. Thus, our results reveal a cellular mechanism whereby growing microtubule plus-ends direct rapid actin assembly. PMID:27199431

  4. Liquid crystal domains and thixotropy of filamentous actin suspensions.

    PubMed

    Kerst, A; Chmielewski, C; Livesay, C; Buxbaum, R E; Heidemann, S R

    1990-06-01

    The thixotropic properties of filamentous actin suspensions were examined by a step-function shearing protocol. Samples of purified filamentous actin were sheared at 0.2 sec-1 in a cone and plate rheometer. We noted a sharp stress overshoot upon the initiation of shear, indicative of a gel state, and a nearly instantaneous drop to zero stress upon cessation of shear. Stress-overshoot recovery was almost complete after 5 min of "rest" before samples were again sheared at 0.2 sec-1. Overshoot recovery increased linearly with the square root of rest time, suggesting that gel-state recovery is diffusion limited. Actin suspensions subjected to oscillatory shearing at frequencies from 0.003 to 30 radians/sec confirmed the existence of a 5-min time scale in the gel, similar to that for stress-overshoot recovery. Flow of filamentous actin was visualized by polarized light observations. Actin from 6 mg/ml to 20 mg/ml showed the "polycrystalline" texture of birefringence typical for liquid crystal structure. At shear rates less than 1 sec-1, flow occurred by the relative movement of irregular, roughly ellipsoidal actin domains 40-140 microns long; the appearance was similar to moving ice floes. At shear rates greater than 1 sec-1, domains decreased in size, possibly by frictional interactions among domains. Eventually domains flow in a "river" of actin aligned by the flow. Our observations confirm our previous domain-friction model for actin rheology. The similarities between the unusual flow properties of actin and cytoplasm argue that cytoplasm also may flow as domains.

  5. Probing the sliding interactions between bundled actin filaments

    NASA Astrophysics Data System (ADS)

    Ward, Andy; Dogic, Zvonimir

    2011-03-01

    Assemblies of filamentous biopolymers are hierarchical materials in which the properties of the overall assemblage are determined by structure and interactions between constituent particles at all hierarchical levels. For example, the overall bending rigidity of a two bundled filaments greatly depends on the bending rigidity of, and the adhesion strength between individual filaments. However, another property of importance is the ability for the filaments to slide freely against one another. Everyday experience indicates that it is much easier to bend a stack of papers in which individual sheets freely slide past each other than the same stack of papers in which all the sheets are irreversibly glued together. Similarly, in filamentous structures the ability for local re-arrangement is of the utmost importance in determining the properties of the structures observed. In order to study this phenomenon we create bundles of biopolymers by inducing attractive interactions between actin filaments via the depletion mechanism. We find that bundles of actin filaments to do not slide freely across one another. In order to characterize these sliding interactions, we perform active experiments using laser tweezers to pull one filament across the other at constant velocity.

  6. MARCKS actin-binding capacity mediates actin filament assembly during mitosis in human hepatic stellate cells.

    PubMed

    Rombouts, Krista; Mello, Tommaso; Liotta, Francesco; Galli, Andrea; Caligiuri, Alessandra; Annunziato, Francesco; Pinzani, Massimo

    2012-08-15

    Cross-linking between the actin cytoskeleton and plasma membrane actin-binding proteins is a key interaction responsible for the mechanical properties of the mitotic cell. Little is known about the identity, the localization, and the function of actin filament-binding proteins during mitosis in human hepatic stellate cells (hHSC). The aim of the present study was to identify and analyze the cross talk between actin and myristoylated alanine-rich kinase C substrate (MARCKS), an important PKC substrate and actin filament-binding protein, during mitosis in primary hHSC. Confocal analysis and chromosomal fraction analysis of mitotic hHSC demonstrated that phosphorylated (P)-MARCKS displays distinct phase-dependent localizations, accumulates at the perichromosomal layer, and is a centrosomal protein belonging to the chromosomal cytosolic fraction. Aurora B kinase (AUBK), an important mitotic regulator, β-actin, and P-MARCKS concentrate at the cytokinetic midbody during cleavage furrow formation. This localization is critical since MARCKS-depletion in hHSC is characterized by a significant loss in cytosolic actin filaments and cortical β-actin that induces cell cycle inhibition and dislocation of AUBK. A depletion of AUBK in hHSC affects cell cycle, resulting in multinucleation. Quantitative live cell imaging demonstrates that the actin filament-binding capacity of MARCKS is key to regulate mitosis since the cell cycle inhibitory effect in MARCKS-depleted cells caused abnormal cell morphology and an aberrant cytokinesis, resulting in a significant increase in cell cycle time. These findings implicate that MARCKS, an important PKC substrate, is essential for proper cytokinesis and that MARCKS and its partner actin are key mitotic regulators during cell cycle in hHSC.

  7. Role of actin filaments in fusopod formation and osteoclastogenesis.

    PubMed

    Wang, Yongqiang; Brooks, Patricia Joyce; Jang, Janet Jinyoung; Silver, Alexandra Shade; Arora, Pamma D; McCulloch, Christopher A; Glogauer, Michael

    2015-07-01

    Cell fusion process is a critical, rate-limiting step in osteoclastogenesis but the mechanisms that regulate fusopod formation are not defined. We characterized fusopod generation in cultured pre-osteoclasts derived from cells stably transfected with a plasmid that expressed a short, actin filament binding peptide (Lifeact) fused to mEGFP that enables localization of actin filaments in living cells. Fusion was initiated at fusopods, which are cell extensions of width >2 μm and that are immunostained for myosin-X at the extension tips. Fusopods formed at the leading edge of larger migrating cells and from the tail of adjacent smaller cells, both of which migrated in the same direction. Staining for DC-STAMP was circumferential and did not localize to cell-cell fusion sites. Compared with wild-type cells, monocytes null for Rac1 exhibited 6-fold fewer fusopods and formed 4-fold fewer multinucleated osteoclasts. From time-lapse images we found that fusion was temporally related to the formation of coherent and spatially isolated bands of actin filaments that originated in cell bodies and extended into the fusopods. These bands of actin filaments were involved in cell fusion after approaching cells formed initial contacts. We conclude that the formation of fusopods is regulated by Rac1 to initiate intercellular contact during osteoclastogenesis. This step is followed by the tightly regulated assembly of bands of actin filaments in fusopods, which lead to closure of the intercellular gap and finally, cell fusion. These novel, actin-dependent processes are important for fusion processes in osteoclastogenesis.

  8. Diffusing wave spectroscopy microrheology of actin filament networks.

    PubMed Central

    Palmer, A; Xu, J; Kuo, S C; Wirtz, D

    1999-01-01

    Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales. PMID:9916038

  9. The dynamics of semiflexible actin filaments in simple shear flow

    NASA Astrophysics Data System (ADS)

    Liu, Yanan; Lindner, Anke; Du Roure, Olivia

    2016-11-01

    The rheological properties of complex fluids made of particles in a suspended fluid depend on the behavior of microscopic particles in flow. A first step to understand this link is to investigate the individual particle dynamics in simple shear flows. A rigid rod will perform so-called Jeffery orbits, however when the rod becomes flexible and Brownian, the behavior in terms of deformation and migration is still to be fully understood. We chose here to address this situation by studying experimentally the behavior of semiflexible polymers. We use actin filaments and combine fluorescent labeling techniques, microfluidic devices to carry out controlled systematical experiments. Different dynamics are observed as a function of the elasto-viscous number, comparing viscous forces to elastic restoring forces ζ = (8 πηγ˙L4) / (LpkB T) . The bending modulus of the actin filaments is given by its persistence length Lp = 17 +/- 1 μm . When increasing the elasto-visous number we subsequently observe tumbling, buckling, and bending under flow. Those observations seem to be in good agreement with recent numerical simulations. At the same time, actin filaments fluctuate due to Brownian motion and these fluctuations can modify the individual dynamics of actin filaments. ERC PaDy No.682367.

  10. Novel actin-like filament structure from Clostridium tetani.

    PubMed

    Popp, David; Narita, Akihiro; Lee, Lin Jie; Ghoshdastider, Umesh; Xue, Bo; Srinivasan, Ramanujam; Balasubramanian, Mohan K; Tanaka, Toshitsugu; Robinson, Robert C

    2012-06-15

    Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines.

  11. Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network

    PubMed Central

    Luo, Weiwei; Lieu, Zi Zhao; Manser, Ed; Bershadsky, Alexander D.; Sheetz, Michael P.

    2016-01-01

    A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. PMID:27760153

  12. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  13. Addition of electrophilic lipids to actin alters filament structure

    SciTech Connect

    Gayarre, Javier; Sanchez, David; Sanchez-Gomez, Francisco J.; Terron, Maria C.; Llorca, Oscar; Perez-Sala, Dolores . E-mail: dperezsala@cib.csic.es

    2006-11-03

    Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-{delta}{sup 12,14}-PGJ{sub 2} (15d-PGJ{sub 2}) and PGA{sub 1} in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA{sub 1} and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ{sub 2} or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ{sub 2} at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.

  14. Addition of electrophilic lipids to actin alters filament structure.

    PubMed

    Gayarre, Javier; Sánchez, David; Sánchez-Gómez, Francisco J; Terrón, María C; Llorca, Oscar; Pérez-Sala, Dolores

    2006-11-03

    Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and PGA(1) in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA(1) and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ(2) or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ(2) at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.

  15. Transportation of nanoscale cargoes by myosin propelled actin filaments.

    PubMed

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A Michael; Månsson, Alf; Kocer, Armagan

    2013-01-01

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached") or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

  16. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites.

    PubMed

    Skillman, Kristen M; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L David

    2011-10-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

  17. Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

    PubMed Central

    Skillman, Kristen M.; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L. David

    2011-01-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility. PMID:21998582

  18. Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

    PubMed Central

    1982-01-01

    Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed. PMID:6890555

  19. Nano-assembly of nanodiamonds by conjugation to actin filaments.

    PubMed

    Bradac, Carlo; Say, Jana M; Rastogi, Ishan D; Cordina, Nicole M; Volz, Thomas; Brown, Louise J

    2016-03-01

    Fluorescent nanodiamonds (NDs) are remarkable objects. They possess unique mechanical and optical properties combined with high surface areas and controllable surface reactivity. They are non-toxic and hence suited for use in biological environments. NDs are also readily available and commercially inexpensive. Here, the exceptional capability of controlling and tailoring their surface chemistry is demonstrated. Small, bright diamond nanocrystals (size ˜30 nm) are conjugated to protein filaments of actin (length ˜3-7 µm). The conjugation to actin filaments is extremely selective and highly target-specific. These unique features, together with the relative simplicity of the conjugation-targeting method, make functionalised nanodiamonds a powerful and versatile platform in biomedicine and quantum nanotechnologies. Applications ranging from using NDs as superior biological markers to, potentially, developing novel bottom-up approaches for the fabrication of hybrid quantum devices that would bridge across the bio/solid-state interface are presented and discussed.

  20. Cortical flow aligns actin filaments to form a furrow

    PubMed Central

    Reymann, Anne-Cecile; Staniscia, Fabio; Erzberger, Anna; Salbreux, Guillaume; Grill, Stephan W

    2016-01-01

    Cytokinesis in eukaryotic cells is often accompanied by actomyosin cortical flow. Over 30 years ago, Borisy and White proposed that cortical flow converging upon the cell equator compresses the actomyosin network to mechanically align actin filaments. However, actin filaments also align via search-and-capture, and to what extent compression by flow or active alignment drive furrow formation remains unclear. Here, we quantify the dynamical organization of actin filaments at the onset of ring assembly in the C. elegans zygote, and provide a framework for determining emergent actomyosin material parameters by the use of active nematic gel theory. We characterize flow-alignment coupling, and verify at a quantitative level that compression by flow drives ring formation. Finally, we find that active alignment enhances but is not required for ring formation. Our work characterizes the physical mechanisms of actomyosin ring formation and highlights the role of flow as a central organizer of actomyosin network architecture. DOI: http://dx.doi.org/10.7554/eLife.17807.001 PMID:27719759

  1. In vitro studies of actin filament and network dynamics

    PubMed Central

    Mullins, R Dyche; Hansen, Scott D

    2013-01-01

    Now that many genomes have been sequenced, a central concern of cell biology is to understand how the proteins they encode work together to create living matter. In vitro studies form an essential part of this program because understanding cellular functions of biological molecules often requires isolating them and reconstituting their activities. In particular, many elements of the actin cytoskeleton were first discovered by biochemical methods and their cellular functions deduced from in vitro experiments. We highlight recent advances that have come from in vitro studies, beginning with studies of actin filaments, and ending with multi-component reconstitutions of complex actin-based processes, including force-generation and cell spreading. We describe both scientific results and the technical innovations that made them possible. PMID:23267766

  2. Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss Physcomitrella patens.

    PubMed

    Yamashita, Hiroko; Sato, Yoshikatsu; Kanegae, Takeshi; Kagawa, Takatoshi; Wada, Masamitsu; Kadota, Akeo

    2011-02-01

    Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.

  3. Force-Velocity Measurements of a Few Growing Actin Filaments

    PubMed Central

    Brangbour, Coraline; du Roure, Olivia; Helfer, Emmanuèle; Démoulin, Damien; Mazurier, Alexis; Fermigier, Marc; Carlier, Marie-France; Bibette, Jérôme; Baudry, Jean

    2011-01-01

    The polymerization of actin in filaments generates forces that play a pivotal role in many cellular processes. We introduce a novel technique to determine the force-velocity relation when a few independent anchored filaments grow between magnetic colloidal particles. When a magnetic field is applied, the colloidal particles assemble into chains under controlled loading or spacing. As the filaments elongate, the beads separate, allowing the force-velocity curve to be precisely measured. In the widely accepted Brownian ratchet model, the transduced force is associated with the slowing down of the on-rate polymerization. Unexpectedly, in our experiments, filaments are shown to grow at the same rate as when they are free in solution. However, as they elongate, filaments are more confined in the interspace between beads. Higher repulsive forces result from this higher confinement, which is associated with a lower entropy. In this mechanism, the production of force is not controlled by the polymerization rate, but is a consequence of the restriction of filaments' orientational fluctuations at their attachment point. PMID:21541364

  4. Actin filaments growing against a barrier with fluctuating shape

    NASA Astrophysics Data System (ADS)

    Sadhu, Raj Kumar; Chatterjee, Sakuntala

    2016-06-01

    We study force generation by a set of parallel actin filaments growing against a nonrigid obstacle, in the presence of an external load. The filaments polymerize by either moving the whole obstacle, with a large energy cost, or by causing local distortion in its shape which costs much less energy. The nonrigid obstacle also has local thermal fluctuations due to which its shape can change with time and we describe this using fluctuations in the height profile of a one-dimensional interface with Kardar-Parisi-Zhang dynamics. We find the shape fluctuations of the barrier strongly affect the force generation mechanism. The qualitative nature of the force-velocity curve is crucially determined by the relative time scale of filament and barrier dynamics. The height profile of the barrier also shows interesting variation with the external load. Our analytical calculations within mean-field theory show reasonable agreement with our simulation results.

  5. Arabidopsis FIM5 decorates apical actin filaments and regulates their organization in the pollen tube

    PubMed Central

    Zhang, Meng; Zhang, Ruihui; Qu, Xiaolu; Huang, Shanjin

    2016-01-01

    The actin cytoskeleton is increasingly recognized as a major regulator of pollen tube growth. Actin filaments have distinct distribution patterns and dynamic properties within different regions of the pollen tube. Apical actin filaments are highly dynamic and crucial for pollen tube growth. However, how apical actin filaments are generated and properly constructed remains an open question. Here we showed that Arabidopsis fimbrin5 (FIM5) decorates filamentous structures throughout the entire tube but is apically concentrated. Apical actin structures are disorganized to different degrees in the pollen tubes of fim5 loss-of-function mutants. Further observations suggest that apical actin structures are not constructed properly because apical actin filaments cannot be maintained at the cortex of fim5 pollen tubes. Actin filaments appeared to be more curved in fim5 pollen tubes and this was confirmed by measurements showing that the convolutedness and the rate of change of convolutedness of actin filaments was significantly increased in fim5 pollen tubes. This suggests that the rigidity of the actin filaments may be compromised in fim5 pollen tubes. Further, the apical cell wall composition is altered, implying that tip-directed vesicle trafficking events are impaired in fim5 pollen tubes. Thus, we found that FIM5 decorates apical actin filaments and regulates their organization in order to drive polarized pollen tube growth. PMID:27117336

  6. SWAP-70 identifies a transitional subset of actin filaments in motile cells.

    PubMed

    Hilpelä, Pirta; Oberbanscheidt, Pia; Hahne, Penelope; Hund, Martin; Kalhammer, Georg; Small, J Victor; Bähler, Martin

    2003-08-01

    Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3'-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actin cytoskeleton as an effector or adaptor protein in response to agonist stimulated phosphatidylinositol (3,4)-bisphosphate production and cell protrusion.

  7. Actin-filament disassembly: it takes two to shrink them fast.

    PubMed

    Winterhoff, Moritz; Faix, Jan

    2015-06-01

    Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level.

  8. CacyBP/SIP as a novel modulator of the thin filament.

    PubMed

    Jurewicz, Ewelina; Ostrowska, Zofia; Jozwiak, Jolanta; Redowicz, Maria Jolanta; Lesniak, Wieslawa; Moraczewska, Joanna; Filipek, Anna

    2013-03-01

    The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin-tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.

  9. Mutation-Specific Effects on Thin Filament Length in Thin Filament Myopathy

    PubMed Central

    de Winter, Josine M.; Joureau, Barbara; Lee, Eun-Jeong; Kiss, Balázs; Yuen, Michaela; Gupta, Vandana A.; Pappas, Christopher T.; Gregorio, Carol C.; Stienen, Ger J. M.; Edvardson, Simon; Wallgren-Pettersson, Carina; Lehtokari, Vilma-Lotta; Pelin, Katarina; Malfatti, Edoardo; Romero, Norma B.; van Engelen, Baziel G.; Voermans, Nicol C.; Donkervoort, Sandra; Bönnemann, C. G.; Clarke, Nigel F.; Beggs, Alan H.; Granzier, Henk; Ottenheijm, Coen A. C.

    2016-01-01

    Objective Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. Methods We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. Results Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force–sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin–thick filament overlap. Interpretation These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. PMID:27074222

  10. Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics

    PubMed Central

    Appaduray, Mark A.; Masedunskas, Andrius; Lucas, Christine A.; Warren, Sean C.; Timpson, Paul; Stear, Jeffrey H.

    2016-01-01

    The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin. PMID:27977753

  11. Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement.

    PubMed

    Wang, Xiu-Ling; Gao, Xin-Qi; Wang, Xue-Chen

    2011-08-01

    Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.

  12. Structural transition of actin filament in a cell-sized water droplet with a phospholipid membrane

    NASA Astrophysics Data System (ADS)

    Hase, M.; Yoshikawa, K.

    2006-03-01

    Actin filament, F-actin, is a semiflexible polymer with a negative charge, and is one of the main constituents of cell membranes. To clarify the effect of cross talk between a phospholipid membrane and actin filaments in cells, we conducted microscopic observations on the structural changes in actin filaments in a cell-sized (several tens of micrometers in diameter) water droplet coated with a phospholipid membrane such as phosphatidylserine (PS; negatively charged head group) or phosphatidylethanolamine (PE; neutral head group) as a simple model of a living cell membrane. With PS, actin filaments are distributed uniformly in the water phase without adsorption onto the membrane surface between 2 and 6mM Mg2+, while between 6 and 12mM Mg2+, actin filaments are adsorbed onto the inner membrane surface. With PE, the actin filaments are uniformly adsorbed onto the inner membrane surface between 2 and 12mM Mg2+. With both PS and PE membranes, at Mg2+ concentrations higher than 12mM, thick bundles are formed in the bulk water droplet accompanied by the dissolution of actin filaments from the membrane surface. The attraction between actin filaments and membrane is attributable to an increase in the translational entropy of counterions accompanied by the adsorption of actin filaments onto the membrane surface. These results suggest that a microscopic water droplet coated with phospholipid can serve as an easy-to-handle model of cell membranes.

  13. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-01-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes. PMID:24058157

  14. Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.

    PubMed

    Li, Hailong; Aksenova, Marina; Bertrand, Sarah J; Mactutus, Charles F; Booze, Rosemarie

    2016-02-10

    Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

  15. Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

    PubMed Central

    Bertrand, Sarah J.; Mactutus, Charles F.; Booze, Rosemarie

    2016-01-01

    Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments. PMID:26889716

  16. Aluminum modifies the viscosity of filamentous actin solutions as measured by optical displacement microviscometry.

    PubMed

    Arnoys, E J; Schindler, M

    2000-01-01

    A microtechnique has been developed that is capable of measuring the viscosity of filamentous actin (F-actin) solutions. This method, called optical displacement microviscometry (ODM), was utilized to determine the changes in viscosity of solutions of rabbit muscle, human platelet, and maize pollen actin when measured in the absence and presence of aluminum. Measurements demonstrated that the viscosity of the different actin solutions decreased with aluminum concentration. In contrast, increases in viscosity were observed when aluminum was added to F-actin solutions containing filamin (chicken gizzard), a protein that bundles actin filaments. Confocal fluorescence imaging of pure actin solutions in the presence of aluminum showed a disrupted actin network composed of fragmented actin filaments in the form of small aggregates. In contrast, in the presence of filamin, aluminum promoted the formation of thicker actin filaments. These measurements demonstrate that aluminum can affect actin filaments differentially depending on the presence of an actin-binding protein. In addition, a strong correlation is observed between the changes in viscosity as measured by ODM and the thickness and assembled state of bundles of actin filaments.

  17. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells

    PubMed Central

    1975-01-01

    The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented. PMID:1202021

  18. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner*

    PubMed Central

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-01-01

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. PMID:26747606

  19. Regulation of structure and function of sarcomeric actin filaments in striated muscle of the nematode Caenorhabditis elegans

    PubMed Central

    Ono, Shoichiro

    2014-01-01

    The nematode Caenorhabditis elegans has been used as a valuable system to study structure and function of striated muscle. The body wall muscle of C. elegans is obliquely striated muscle with highly organized sarcomeric assembly of actin, myosin, and other accessary proteins. Genetic and molecular biological studies in C. elegans have identified a number of genes encoding structural and regulatory components for the muscle contractile apparatuses, and many of them have counterparts in mammalian cardiac and skeletal muscles or striated muscles in other invertebrates. Applicability of genetics, cell biology, and biochemistry has made C. elegans an excellent system to study mechanisms of muscle contractility and assembly and maintenance of myofibrils. This review focuses on the regulatory mechanisms of structure and function of actin filaments in the C. elegans body wall muscle. Sarcomeric actin filaments in C. elegans muscle are associated with the troponin-tropomyosin system that regulates the actin-myosin interaction. Proteins that bind to the side and ends of actin filaments support ordered assembly of thin filaments. Furthermore, regulators of actin dynamics play important roles in initial assembly, growth, and maintenance of sarcomeres. The knowledge acquired in C. elegans can serve as bases to understand the basic mechanisms of muscle structure and function. PMID:25125169

  20. Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.

    PubMed

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament-severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca(2+), in vitro. Analysis of a mutant that bears a point mutation at the Ca(2+) binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca(2+) level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament-severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.

  1. Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments

    PubMed Central

    1995-01-01

    The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin- binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere. PMID:7822423

  2. Bacterial actin MreB forms antiparallel double filaments

    PubMed Central

    van den Ent, Fusinita; Izoré, Thierry; Bharat, Tanmay AM; Johnson, Christopher M; Löwe, Jan

    2014-01-01

    Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments. DOI: http://dx.doi.org/10.7554/eLife.02634.001 PMID:24843005

  3. Site-specific cation release drives actin filament severing by vertebrate cofilin

    PubMed Central

    Kang, Hyeran; Bradley, Michael J.; Cao, Wenxiang; Zhou, Kaifeng; Grintsevich, Elena E.; Michelot, Alphée; Sindelar, Charles V.; Hochstrasser, Mark; De La Cruz, Enrique M.

    2014-01-01

    Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this “stiffness cation” unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics. PMID:25468977

  4. Mammalian CARMIL Inhibits Actin Filament Capping by Capping Protein

    PubMed Central

    Yang, Changsong; Pring, Martin; Wear, Martin A.; Huang, Minzhou; Cooper, John A.; Svitkina, Tatyana M.; Zigmond, Sally H.

    2009-01-01

    Summary Actin polymerization in cells occurs via filament elongation at the barbed end. Proteins that cap the barbed end terminate this elongation. Heterodimeric capping protein (CP) is an abundant and ubiquitous protein that caps the barbed end. We find that the mouse homolog of the adaptor protein CARMIL (mCARMIL) binds CP with high affinity and decreases its affinity for the barbed end. Addition of mCARMIL to cell extracts increases the rate and extent of Arp2/3 or spectrin-actin seed-induced polymerization. In cells, GFP-mCARMIL concentrates in lamellipodia and increases the fraction of cells with large lamellipodia. Decreasing mCARMIL levels by siRNA transfection lowers theF-actin level and slows cell migration through a mechanism that includes decreased lamellipodia protrusion. This phenotype is reversed by full-length mCARMIL but not mCARMIL lacking the domain that binds CP. Thus, mCARMIL is a key regulator of CP and has profound effects on cell behavior. PMID:16054028

  5. Fetal akinesia caused by a novel actin filament aggregate myopathy skeletal muscle actin gene (ACTA1) mutation.

    PubMed

    Stenzel, Werner; Prokop, Stefan; Kress, Wolfram; Huppmann, Stephanie; Loui, Andrea; Sarioglu, Nanette M E; Laing, Nigel G; Sparrow, John C; Heppner, Frank L; Goebel, Hans H

    2010-08-01

    We report a female newborn, diagnosed with fetal akinesia in utero, who died one hour after birth. Post-mortem muscle biopsy demonstrated actin-filament myopathy based on immunolabelling for sarcomeric actin, and large areas of filaments, without rod formation, ultrastructurally. Analysis of DNA extracted from the muscle disclosed a novel de novo heterozygous c.44G>A, GGC>GAC, 'p.Gly15Asp' mutation in the ACTA1 gene. Analysis of the location of the mutated amino-acid in the actin molecule suggests the mutation most likely causes abnormal nucleotide binding, and consequent pathological actin polymerization. This case emphasizes the association of fetal akinesia with actin-filament myopathy.

  6. Thin Filament-Reconstituted Skinned Muscle Fibers for the Study of Muscle Physiology

    PubMed Central

    Higuchi, Sayaka; Tsukasaki, Yoshikazu; Fukuda, Norio; Kurihara, Satoshi; Fujita, Hideaki

    2011-01-01

    We review the use of thin filament-reconstituted muscle fibers in the study of muscle physiology. Thin filament extraction and reconstitution protocol is a powerful technique to study the role of each component of the thin filament. It is also useful for studying the properties of genetically modified molecules such as actin and tropomyosin. We also review the combination of this protocol with sinusoidal analysis, which will provide a solid technique for determining the effect of regulatory proteins on actomyosin interaction and concomitant cross-bridge kinetics. We suggest that thin filament-reconstituted muscle fibers are an ideal system for studying muscle physiology especially when gene modifications of actin or tropomyosin are involved. PMID:22131807

  7. Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

    PubMed

    Pang, K M; Lee, E; Knecht, D A

    1998-03-26

    Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

  8. Fission yeast IQGAP arranges actin filaments into the cytokinetic contractile ring

    PubMed Central

    Takaine, Masak; Numata, Osamu; Nakano, Kentaro

    2009-01-01

    The contractile ring (CR) consists of bundled actin filaments and myosin II; however, the actin-bundling factor remains elusive. We show that the fission yeast Schizosaccharomyces pombe IQGAP Rng2 is involved in the generation of CR F-actin and required for its arrangement into a ring. An N-terminal fragment of Rng2 is necessary for the function of Rng2 and is localized to CR F-actin. In vitro the fragment promotes actin polymerization and forms linear arrays of F-actin, which are resistant to the depolymerization induced by the actin-depolymerizing factor Adf1. Our findings indicate that Rng2 is involved in the generation of CR F-actin and simultaneously bundles the filaments and regulates its dynamics by counteracting the effects of Adf1, thus enabling the reconstruction of CR F-actin bundles, which provides an insight into the physical properties of the building blocks that comprise the CR. PMID:19713940

  9. Preparation of filamentous actin for polarized total internal reflection fluorescence microscopy (polTIRFM) motility assays.

    PubMed

    Beausang, John F; Sun, Yujie; Quinlan, Margot E; Forkey, Joseph N; Goldman, Yale E

    2012-05-01

    Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. In this protocol, filamentous actin (F-actin) is polymerized from purified, monomeric actin (G-actin) for use in polTIRFM motility assays in which actin interacts with myosin. The procedures include (1) the preparation of unlabeled F-actin from G-actin; (2) the preparation of F-actin that is sparsely labeled with 6'-IATR (6'-iodoacetamidotetramethylrhodamine); and (3) the preparation of F-actin with a combination of unlabeled, biotinylated, and rhodamine-labeled monomers. Rhodamine-phalloidin actin, also used in polTIRFM assays, can be prepared using a procedure similar to the one for unlabeled actin.

  10. The ATP binding cassette transporter, ABCG1, localizes to cortical actin filaments

    PubMed Central

    Pandzic, Elvis; Gelissen, Ingrid C.; Whan, Renee; Barter, Philip J.; Sviridov, Dmitri; Gaus, Katharina; Rye, Kerry-Anne; Cochran, Blake J.

    2017-01-01

    The ATP-binding cassette sub-family G member 1 (ABCG1) exports cellular cholesterol to high-density lipoproteins (HDL). However, a number of recent studies have suggested ABCG1 is predominantly localised to intracellular membranes. In this study, we found that ABCG1 was organized into two distinct cellular pools: one at the plasma membrane and the other associated with the endoplasmic reticulum (ER). The plasma membrane fraction was organized into filamentous structures that were associated with cortical actin filaments. Inhibition of actin polymerization resulted in complete disruption of ABCG1 filaments. Cholesterol loading of the cells increased the formation of the filamentous ABCG1, the proximity of filamentous ABCG1 to actin filaments and the diffusion rate of membrane associated ABCG1. Our findings suggest that the actin cytoskeleton plays a critical role in the plasma membrane localization of ABCG1. PMID:28165022

  11. Regional orientation of actin filaments in the pericanalicular cytoplasm of rat hepatocytes.

    PubMed

    Ishii, M; Washioka, H; Tonosaki, A; Toyota, T

    1991-12-01

    To elucidate how actin filaments participate in bile formation, polarity of actin filaments in the pericanalicular cytoplasm was determined with myosin subfragment 1 by transmission electron microscopy of ultrathin sections and deep-etching replicas. Densely concentrated actin filaments were identified around the bile canaliculi in the forms of microvillous core filaments, pericanalicular web filaments, and filaments on the junctional complex. They bound subfragment 1 to form double-helical strands on the deep-etching replica or typical arrowheads on the ultrathin section. All microvillous core filaments showed their arrowheads pointing basally, suggesting the molecular growth occurring at their apical ends. In contrast, filaments of the pericanalicular web, running in parallel to the cell surface, showed unfixed polarities as indicated by their arrowheads. Furthermore, neighboring filament pairs often showed opposite polarities, an alignment necessary for filament sliding. The junctional complex had filaments with arrowheads pointed mostly at the cell center with a small number in opposite direction. In addition, a group of sporadic filaments appeared to be installed to link to both the canalicular membrane and coated vesicles. Such regionally specialized actin filaments are considered inclusively to form a cytoskeletal system that is in charge of (a) maintenance of length of the microvilli, (b) contraction of the canalicular walls, and (c) translocation of coated vesicles in the pericanalicular cytoplasm.

  12. Structural complexity of filaments formed from the actin and tubulin folds

    PubMed Central

    Jiang, Shimin; Ghoshdastider, Umesh; Narita, Akihiro; Popp, David

    2016-01-01

    ABSTRACT From yeast to man, an evolutionary distance of 1.3 billion years, the F-actin filament structure has been conserved largely in line with the 94% sequence identity. The situation is entirely different in bacteria. In comparison to eukaryotic actins, the bacterial actin-like proteins (ALPs) show medium to low levels of sequence identity. This is extreme in the case of the ParM family of proteins, which often display less than 20% identity. ParMs are plasmid segregation proteins that form the polymerizing motors that propel pairs of plasmids to the extremities of a cell prior to cell division, ensuring faithful inheritance of the plasmid. Recently, exotic ParM filament structures have been elucidated that show ParM filament geometries are not limited to the standard polar pair of strands typified by actin. Four-stranded non-polar ParM filaments existing as open or closed nanotubules are found in Clostridium tetani and Bacillus thuringiensis, respectively. These diverse architectures indicate that the actin fold is capable of forming a large variety of filament morphologies, and that the conception of the “actin” filament has been heavily influenced by its conservation in eukaryotes. Here, we review the history of the structure determination of the eukaryotic actin filament to give a sense of context for the discovery of the new ParM filament structures. We describe the novel ParM geometries and predict that even more complex actin-like filaments may exist in bacteria. Finally, we compare the architectures of filaments arising from the actin and tubulin folds and conclude that the basic units possess similar properties that can each form a range of structures. Thus, the use of the actin fold in microfilaments and the tubulin fold for microtubules likely arose from a wider range of filament possibilities, but became entrenched as those architectures in early eukaryotes. PMID:28042378

  13. Phosphatidylserine liposomes can be tethered by caldesmon to actin filaments.

    PubMed Central

    Makuch, R; Zasada, A; Mabuchi, K; Krauze, K; Wang, C L; Dabrowska, R

    1997-01-01

    Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes. Images FIGURE 2 FIGURE 4 FIGURE 6 PMID:9284327

  14. Three-dimensional organization of troponin on cardiac muscle thin filaments in the relaxed state.

    PubMed

    Yang, Shixin; Barbu-Tudoran, Lucian; Orzechowski, Marek; Craig, Roger; Trinick, John; White, Howard; Lehman, William

    2014-02-18

    Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca(2+) sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.

  15. Profilin-Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis1[OPEN

    PubMed Central

    Cao, Lingyan; Blanchoin, Laurent; Staiger, Christopher J.

    2016-01-01

    Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion. PMID:26574597

  16. Profilin Interaction with Actin Filament Barbed End Controls Dynamic Instability, Capping, Branching, and Motility

    PubMed Central

    Pernier, Julien; Shekhar, Shashank; Jegou, Antoine; Guichard, Bérengère; Carlier, Marie-France

    2016-01-01

    Summary Cell motility and actin homeostasis depend on the control of polarized growth of actin filaments. Profilin, an abundant regulator of actin dynamics, supports filament assembly at barbed ends by binding G-actin. Here, we demonstrate how, by binding and destabilizing filament barbed ends at physiological concentrations, profilin also controls motility, cell migration, and actin homeostasis. Profilin enhances filament length fluctuations. Profilin competes with Capping Protein at barbed ends, which generates a lower amount of profilin-actin than expected if barbed ends were tightly capped. Profilin competes with barbed end polymerases, such as formins and VopF, and inhibits filament branching by WASP-Arp2/3 complex by competition for filament barbed ends, accounting for its as-yet-unknown effects on motility and metastatic cell migration observed in this concentration range. In conclusion, profilin is a major coordinator of polarized growth of actin filaments, controlled by competition between barbed end cappers, trackers, destabilizers, and filament branching machineries. PMID:26812019

  17. Actin- and myosin-like filaments in rat brain pericytes.

    PubMed

    Le Beux, Y J; Willemot, J

    1978-04-01

    Heavy meromyosin (HMM) labeling was used to identify the nature of the filaments which form bundles in the cytoplasm of the pericytes in brain tissue. Rat brain tissue pieces were incubated in glycerol solutions at 4 degrees and then transferred into buffer (pH 7.0), (1) without HMM, (2) with HMM, (3) with HMM + 5 mM ATP, and (4) with HMM + 2.5 mM Na+ pyrophosphate. In pericytes from untreated tissue, smooth-surfaced microfilaments, averaging 6 nm in diameter, appear to branch and anastomose and to anchor on the plasma membrane. After exposure to HMM, the number and the density of the microfilaments are strikingly increased. These tightly-packed microfilaments are now heavily coated with exogeneous HMM thus increasing in width to 18-20 mm. They intertwine in closely-woven networks. After incubation in HMM solutions containing ATP or Na+ phosphate, they are no longer coated with thick sidearms. It can thus be concluded that these microfilaments are of actin-like nature. In addition, after incubation in ATP, they are intermingled with, and converge onto the surfaces of, thick, tapered filaments, which we have tentatively identified as of myosin-like nature. Thus, it appears that certain of the major elements necessary for contraction are present in brain pericytes.

  18. Toxoplasma gondii profilin acts primarily to sequester G-actin while formins efficiently nucleate actin filament formation in vitro.

    PubMed

    Skillman, Kristen M; Daher, Wassim; Ma, Christopher I; Soldati-Favre, Dominique; Sibley, L David

    2012-03-27

    Apicomplexan parasites employ gliding motility that depends on the polymerization of parasite actin filaments for host cell entry. Despite this requirement, parasite actin remains almost entirely unpolymerized at steady state; formation of filaments required for motility relies on a small repertoire of actin-binding proteins. Previous studies have shown that apicomplexan formins and profilin exhibit canonical functions on heterologous actins from higher eukaryotes; however, their biochemical properties on parasite actins are unknown. We therefore analyzed the impact of T. gondii profilin (TgPRF) and FH1-FH2 domains of two formin isoforms in T. gondii (TgFRM1 and TgFRM2) on the polymerization of T. gondii actin (TgACTI). Our findings based on in vitro assays demonstrate that TgFRM1-FH1-FH2 and TgFRM2-FH1-FH2 dramatically enhanced TgACTI polymerization in the absence of profilin, making them the sole protein factors known to initiate polymerization of this normally unstable actin. In addition, T. gondii formin domains were shown to both initiate polymerization and induce bundling of TgACTI filaments; however, they did not rely on TgPRF for these activities. In contrast, TgPRF sequestered TgACTI monomers, thus inhibiting polymerization even in the presence of formins. Collectively, these findings provide insight into the unusual control mechanisms of actin dynamics within the parasite.

  19. Covalent attachment of actin filaments to Tween 80 coated polystyrene beads for cargo transportation.

    PubMed

    Kaur, Harsimran; Das, Tapan; Kumar, Rajesh; Ajore, Ram; Bharadwaj, Lalit M

    2008-04-01

    In this manuscript, a new strategy has been reported for circumscribed covalent attachment of barbed and pointed ends of actin filaments to polystyrene beads. A comparative study of attachment of actin filaments to polystyrene beads was performed by blocking functionally active sites on polystyrene beads with nonionic detergents such as Tween 20, Tween 80 and polyethylene glycol (PEG). Effective blocking of active sites was obtained with Tween 80 at 0.1% concentration. Attachment of single bundle of actin filament to bead was assessed by rotational motion of bead tailed actin in front and lateral view. Velocity of actin filaments attached to different size of beads in in-vitro motility assay was calculated to ascertain their attachments. Velocity of actin attached to 1.0 and 3.0 microm polystyrene beads was reduced to 3.0-4.0 and 0.0-1.0 microm/s, respectively as compared to free actin velocity of 4.0-6.0 microm/s. Single point attachment of actin filaments to different size of beads was assessed by decrease in sliding velocity. Present study provides insight into the actin-myosin based molecular motor systems for drug delivery and biosensors applications.

  20. Tracer diffusion through F-actin: effect of filament length and cross-linking.

    PubMed Central

    Jones, J D; Luby-Phelps, K

    1996-01-01

    We have determined diffusion coefficients for small (50- to 70-nm diameter) fluorescein-thiocarbamoyl-labeled Ficoll tracers through F-actin as a function of filament length and cross-linking. fx45 was used to regulate filament length and avidin/biotinylated actin or ABP-280 was used to prepare cross-linked actin gels. We found that tracer diffusion was generally independent of filament length in agreement with theoretical predictions for diffusion through solutions of rods. However, in some experiments diffusion was slower through short (< or = 1.0 micron) filaments, although this result was not consistently reproducible. Measured diffusion coefficients through unregulated F-actin and filaments of lengths > 1.0 micron were more rapid than predicted by theory for tracer diffusion through rigid, random networks, which was consistent with some degree of actin bundling. Avidin-induced cross-linking of biotinylated F-actin did not affect diffusion through unregulated F-actin, but in cases where diffusion was slower through short filaments this cross-linking method resulted in enhanced tracer diffusion rates indistinguishable from unregulated F-actin. This finding, in conjunction with increased turbidity of 1.0-micron filaments upon avidin cross-linking, indicated that this cross-linking method induces F-actin bundling. By contrast, ABP-280 cross-linking retarded diffusion through unregulated F-actin and decreased turbidity. Tracer diffusion under these conditions was well approximated by the diffusion theory. Both cross-linking procedures resulted in gel formation as determined by falling ball viscometry. These results demonstrate that network microscopic geometry is dependent on the cross-linking method, although both methods markedly increase F-actin macroscopic viscosity. PMID:8913611

  1. Viral infectivity and intracellular distribution of matrix (M) protein of canine distemper virus are affected by actin filaments.

    PubMed

    Klauschies, F; Gützkow, T; Hinkelmann, S; von Messling, V; Vaske, B; Herrler, G; Haas, L

    2010-09-01

    To investigate the role of cytoskeletal components in canine distemper virus (CDV) replication, various agents were used that interfere with turnover of actin filaments and microtubules. Only inhibition of actin filaments significantly reduced viral infectivity. Analysis of the intracellular localization of the viral matrix (M) protein revealed that it aligned along actin filaments. Treatment with actin filament-disrupting drugs led to a marked intracellular redistribution of M protein during infection as well as transfection. In contrast, the localization of the CDV fusion (F) protein was not significantly changed during transfection. Thus, a M protein-actin filament interaction appears to be important for generation of infectious CDV.

  2. Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization.

    PubMed

    Xue, Bo; Leyrat, Cedric; Grimes, Jonathan M; Robinson, Robert C

    2014-10-28

    Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation.

  3. Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks.

    PubMed

    Christensen, Jenna R; Hocky, Glen M; Homa, Kaitlin E; Morganthaler, Alisha N; Hitchcock-DeGregori, Sarah E; Voth, Gregory A; Kovar, David R

    2017-03-10

    The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.

  4. Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation

    PubMed Central

    Yamagishi, Yuka; Abe, Hiroshi

    2015-01-01

    We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation. PMID:26424802

  5. Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity

    PubMed Central

    1992-01-01

    The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video- enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three- dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament

  6. Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV⃞

    PubMed Central

    Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

    2001-01-01

    An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIα impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIα contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors. PMID:11739797

  7. F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling

    PubMed Central

    1995-01-01

    Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product). PMID:7622563

  8. Possible association of actin filaments with chloroplasts of spinach mesophyll cells in vivo and in vitro.

    PubMed

    Kumatani, T; Sakurai-Ozato, N; Miyawaki, N; Yokota, E; Shimmen, T; Terashima, I; Takagi, S

    2006-11-01

    In palisade mesophyll cells of spinach (Spinacia oleracea L.) kept under low-intensity white light, chloroplasts were apparently immobile and seemed to be surrounded by fine bundles of actin filaments. High-intensity blue light induced actin-dependent chloroplast movement concomitant with the appearance of a couple of long, straight bundles of actin filaments in each cell, whereas high-intensity red light was essentially ineffective in inducing these responses. The actin organization observed under low-intensity white light has been postulated to function in anchoring chloroplasts at proper intracellular positions through direct interaction with the chloroplasts. Intact chloroplasts, which retained their outer envelopes, were isolated after homogenization of leaves and Percoll centrifugation. No endogenous actin was detected by immunoblotting in the final intact-chloroplast fraction prepared from the leaves kept under low-intensity white light or in darkness. In cosedimentation assays with exogenously added skeletal muscle filamentous actin, however, actin was detected in the intact-chloroplast fraction precipitated after low-speed centrifugation. The association of actin with chloroplasts was apparently dependent on incubation time and chloroplast density. After partial disruption of the outer envelope of isolated chloroplasts by treatment with trypsin, actin was no longer coprecipitated. The results suggest that chloroplasts in spinach leaves can directly interact with actin, and that this interaction may be involved in the regulation of intracellular positioning of chloroplasts.

  9. Troponin-like regulation in muscle thin filaments of the mussel Crenomytilus grayanus (Bivalvia: Mytiloida).

    PubMed

    Vyatchin, Ilya G; Shevchenko, Ulyana V; Lazarev, Stanislav S; Matusovsky, Oleg S; Shelud'ko, Nikolay S

    2015-10-01

    Muscles of bivalve molluscs have double calcium regulation--myosin-linked and actin-linked. While the mechanism of myosin-linked regulation is sufficiently studied, there is still no consensus on the mechanism of actin-linked regulation. Earlier we showed a high degree of Ca2+-sensitivity of thin filaments from the adductor muscle of the mussel Crenomytilus grayanus (Mytiloida). In order to elucidate the nature of this regulation, we isolated the fraction of minor proteins from the mussel thin filaments, which confers Ca2+-sensitivity to reconstituted actomyosin-tropomyosin. Proteins of this fraction, ABP-19, ABP-20, and ABP-28, were chromatographically purified and identified. According to the results of mass spectrometry and Western blot analysis, as well as by their functional properties, these mussel actin-binding proteins appeared to correspond to the troponin components from the skeletal muscles of vertebrates (TnC, TnI and TnT). The reconstituted mussel troponin complex confers to actomyosin-tropomyosin more than 80% Ca2+-sensitivity. The in vivo molar ratio of actin/tropomyosin/troponin was calculated to be 7:1:0.5, i.e., the content of troponin in mussel thin filaments is two times lower than in thin filaments of skeletal muscles of vertebrates. These data demonstrate that troponin-like regulation found in the catch muscle of the mussel C. grayanus is present at least in two suborders of bivalves: Pectinoida and Mytiloida.

  10. Quantitative Analysis of Filament Branch Orientation in Listeria Actin Comet Tails.

    PubMed

    Jasnin, Marion; Crevenna, Alvaro H

    2016-02-23

    Several bacterial and viral pathogens hijack the host actin cytoskeleton machinery to facilitate spread and infection. In particular, Listeria uses Arp2/3-mediated actin filament nucleation at the bacterial surface to generate a branched network that will help propel the bacteria. However, the mechanism of force generation remains elusive due to the lack of high-resolution three-dimensional structural data on the spatial organization of the actin mother and daughter (i.e., branch) filaments within this network. Here, we have explored the three-dimensional structure of Listeria actin tails in Xenopus laevis egg extracts using cryo-electron tomography. We found that the architecture of Listeria actin tails is shared between those formed in cells and in cell extracts. Both contained nanoscopic bundles along the plane of the substrate, where the bacterium lies, and upright filaments (also called Z filaments), both oriented tangentially to the bacterial cell wall. Here, we were able to identify actin filament intersections, which likely correspond to branches, within the tails. A quantitative analysis of putative Arp2/3-mediated branches in the actin network showed that mother filaments lie on the plane of the substrate, whereas daughter filaments have random deviations out of this plane. Moreover, the analysis revealed that branches are randomly oriented with respect to the bacterial surface. Therefore, the actin filament network does not push directly toward the surface but rather accumulates, building up stress around the Listeria surface. Our results favor a mechanism of force generation for Listeria movement where the stress is released into propulsive motion.

  11. Quantitative Analysis of Filament Branch Orientation in Listeria Actin Comet Tails

    PubMed Central

    Jasnin, Marion; Crevenna, Alvaro H.

    2016-01-01

    Several bacterial and viral pathogens hijack the host actin cytoskeleton machinery to facilitate spread and infection. In particular, Listeria uses Arp2/3-mediated actin filament nucleation at the bacterial surface to generate a branched network that will help propel the bacteria. However, the mechanism of force generation remains elusive due to the lack of high-resolution three-dimensional structural data on the spatial organization of the actin mother and daughter (i.e., branch) filaments within this network. Here, we have explored the three-dimensional structure of Listeria actin tails in Xenopus laevis egg extracts using cryo-electron tomography. We found that the architecture of Listeria actin tails is shared between those formed in cells and in cell extracts. Both contained nanoscopic bundles along the plane of the substrate, where the bacterium lies, and upright filaments (also called Z filaments), both oriented tangentially to the bacterial cell wall. Here, we were able to identify actin filament intersections, which likely correspond to branches, within the tails. A quantitative analysis of putative Arp2/3-mediated branches in the actin network showed that mother filaments lie on the plane of the substrate, whereas daughter filaments have random deviations out of this plane. Moreover, the analysis revealed that branches are randomly oriented with respect to the bacterial surface. Therefore, the actin filament network does not push directly toward the surface but rather accumulates, building up stress around the Listeria surface. Our results favor a mechanism of force generation for Listeria movement where the stress is released into propulsive motion. PMID:26497103

  12. High-speed depolymerization at actin filament ends jointly catalysed by Twinfilin and Srv2/CAP.

    PubMed

    Johnston, Adam B; Collins, Agnieszka; Goode, Bruce L

    2015-11-01

    Purified actin filaments depolymerize slowly, and cytosolic conditions strongly favour actin assembly over disassembly, which has left our understanding of how actin filaments are rapidly turned over in vivo incomplete. One mechanism for driving filament disassembly is severing by factors such as Cofilin. However, even after severing, pointed-end depolymerization remains slow and unable to fully account for observed rates of actin filament turnover in vivo. Here we describe a mechanism by which Twinfilin and Cyclase-associated protein work in concert to accelerate depolymerization of actin filaments by 3-fold and 17-fold at their barbed and pointed ends, respectively. This mechanism occurs even under assembly conditions, allowing reconstitution and direct visualization of individual filaments undergoing tunable, accelerated treadmilling. Further, we use specific mutations to demonstrate that this activity is critical for Twinfilin function in vivo. These findings fill a major gap in our knowledge of cellular disassembly mechanisms, and suggest that depolymerization and severing may be deployed separately or together to control the dynamics and architecture of distinct actin networks.

  13. High Speed Depolymerization at Actin Filament Ends Jointly Catalyzed by Twinfilin and Srv2/CAP

    PubMed Central

    Johnston, Adam B.; Collins, Agnieszka; Goode, Bruce L.

    2015-01-01

    Purified actin filaments depolymerize slowly, and cytosolic conditions strongly favor actin assembly over disassembly, which has left our understanding of how actin filaments are rapidly turned over in vivo incomplete 1,2. One mechanism for driving filament disassembly is severing by factors such as Cofilin. However, even after severing, pointed end depolymerization remains slow and unable to fully account for observed rates of actin filament turnover in vivo. Here we describe a mechanism by which Twinfilin and Cyclase-associated protein work in concert to accelerate depolymerization of actin filaments by 3-fold and 17-fold at their barbed and pointed ends, respectively. This mechanism occurs even under assembly conditions, allowing reconstitution and direct visualization of individual filaments undergoing tunable, accelerated treadmilling. Further, we use specific mutations to demonstrate that this activity is critical for Twinfilin function in vivo. These findings fill a major gap in our knowledge of mechanisms, and suggest that depolymerization and severing may be deployed separately or together to control the dynamics and architecture of distinct actin networks. PMID:26458246

  14. The effects of formins on the conformation of subdomain 1 in actin filaments.

    PubMed

    Ujfalusi, Zoltán; Barkó, Szilvia; Hild, Gábor; Nyitrai, Miklós

    2010-01-21

    In this study we investigated the effects of formins on the conformation of actin filaments by using the method of fluorescence quenching. Actin was labelled with IAEDANS at Cys(374) and the quencher was acrylamide. The results showed that formin binding induced structural changes in the subdomain 1 of actin protomers which were reflected by greater quenching constants (K(SV)). Simultaneously the fraction of the fluorophore population accessible for the quencher (alpha) decreased. These observations suggest that the conformational distribution characteristic for the actin protomers became broader after the binding of formins, for which the structural framework was provided by a more flexible protein matrix in the microenvironment of the label. The effects of formins depended on the formin:actin molar ratio, and also on the ionic strength of the medium. These observations are in agreement with previous results and underline the importance of the intramolecular conformational changes induced by formins in the structure of actin filaments.

  15. ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays

    PubMed Central

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M.; Lowe, Martin; Vartiainen, Maria K.; Jokitalo, Eija

    2014-01-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. PMID:24523293

  16. Simultaneous quantification of actin monomer and filament dynamics with modeling-assisted analysis of photoactivation

    PubMed Central

    Kapustina, Maryna; Read, Tracy-Ann

    2016-01-01

    ABSTRACT Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments. PMID:27831495

  17. ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays.

    PubMed

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M; Lowe, Martin; Vartiainen, Maria K; Jokitalo, Eija

    2014-04-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

  18. Actin-crosslinking protein regulation of filament movement in motility assays: a theoretical model.

    PubMed Central

    Janson, L W; Taylor, D L

    1994-01-01

    The interaction of single actin filaments on a myosin-coated coverslip has been modeled by several authors. One model adds a component of "frictional drag" by myosin heads that oppose movement of the actin filaments. We have extended this concept by including the resistive drag from actin crosslinking proteins to understand better the relationship among crosslinking number, actin-myosin force generation, and motility. The validity of this model is supported by agreement with the experimental results from a previous study in which crosslinking proteins were added with myosin molecules under otherwise standard motility assay conditions. The theoretical relationship provides a means to determine many physical parameters that characterize the interaction between a single actin filament and a single actin-crosslinking molecule (various types). In particular, the force constant of a single filamin molecule is calculated as 1.105 pN, approximately 3 times less than a driving myosin head (3.4 pN). Knowledge of this parameter and others derived from this model allows a better understanding of the interaction between myosin and the actin/actin-binding protein cytoskeleton and the role of actin-binding proteins in the regulation and modulation of motility. PMID:7811954

  19. Quasiperiodic distribution of rigor cross-bridges along a reconstituted thin filament in a skeletal myofibril.

    PubMed

    Suzuki, Madoka; Ishiwata, Shin'ichi

    2011-12-07

    Electron microscopy has shown that cross-bridges (CBs) are formed at the target zone that is periodically distributed on the thin filament in striated muscle. Here, by manipulating a single bead-tailed actin filament with optical tweezers, we measured the unbinding events of rigor CBs one by one on the surface of the A-band in rabbit skeletal myofibrils. We found that the spacings between adjacent CBs were not always the same, and instead were 36, 72, or 108 nm. Tropomyosin and troponin did not affect the CB spacing except for a relative increase in the appearance of longer spacing in the presence of Ca(2+). In addition, in an in vitro assay where myosin molecules were randomly distributed, were obtained the same spacing, i.e., a multiple of 36 nm. These results indicate that the one-dimensional distribution of CBs matches with the 36-nm half pitch of a long helical structure of actin filaments. A stereospecific model composed of three actin protomers per target zone was shown to explain the experimental results. Additionally, the unbinding force (i.e., the binding affinity) of CBs for the reconstituted thin filaments was found to be larger and smaller relative to that for actin filaments with and without Ca(2+), respectively.

  20. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

    PubMed

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q P; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q P

    2016-10-20

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

  1. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

    PubMed Central

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T.; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q. P.; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q. P.

    2016-01-01

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells. PMID:27762277

  2. The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model

    PubMed Central

    1991-01-01

    Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F- actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament, and a slender "tip" defining its "pointed" end (i.e., relative to the "arrowhead" pattern revealed after stoichiometric decoration of the filaments with myosin subfragment 1). Concavities running approximately parallel to the filament axis both on the inner and outer face of the subunit define a distinct cleft separating the subunit into two domains of similar size: an inner domain confined to radii less than or equal to 2.5-nm forms the uninterrupted backbone of the two long-pitch helical strands, and an outer domain placed at radii of 2-5-nm protrudes radially and thus predominantly

  3. Distribution pattern changes of actin filaments during chloroplast movement in Adiantum capillus-veneris.

    PubMed

    Tsuboi, Hidenori; Wada, Masamitsu

    2012-05-01

    Chloroplasts change their positions in a cell in response to light intensities. The photoreceptors involved in chloroplast photo-relocation movements and the behavior of chloroplasts during their migration were identified in our previous studies, but the mechanism of movement has yet to be clarified. In this study, the behavior of actin filaments under various light conditions was observed in Adiantum capillus-veneris gametophytes. In chloroplasts staying in one place under a weak light condition and not moving, circular structures composed of actin filaments were observed around the chloroplast periphery. In contrast, short actin filaments were observed at the leading edge of moving chloroplasts induced by partial cell irradiation. In the dark, the circular structures found under the weak light condition disappeared and then reappeared around the moving chloroplasts. Mutant analyses revealed that the disappearance of the circular actin structure was mediated by the blue light photoreceptor, phototropin2.

  4. The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

    PubMed Central

    Bane, Kartik S.; Singer, Mirko; Reinig, Miriam; Klug, Dennis; Heiss, Kirsten; Baum, Jake; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics. PMID:27409081

  5. Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport

    PubMed Central

    Kumar, Saroj; ten Siethoff, Lasse; Persson, Malin; Lard, Mercy; te Kronnie, Geertruy; Linke, Heiner; Månsson, Alf

    2012-01-01

    Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm−1). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments. PMID:23056279

  6. The Drosophila formin Fhos is a primary mediator of sarcomeric thin-filament array assembly

    PubMed Central

    Shwartz, Arkadi; Dhanyasi, Nagaraju; Schejter, Eyal D; Shilo, Ben-Zion

    2016-01-01

    Actin-based thin filament arrays constitute a fundamental core component of muscle sarcomeres. We have used formation of the Drosophila indirect flight musculature for studying the assembly and maturation of thin-filament arrays in a skeletal muscle model system. Employing GFP-tagged actin monomer incorporation, we identify several distinct phases in the dynamic construction of thin-filament arrays. This sequence includes assembly of nascent arrays after an initial period of intensive microfilament synthesis, followed by array elongation, primarily from filament pointed-ends, radial growth of the arrays via recruitment of peripheral filaments and continuous barbed-end turnover. Using genetic approaches we have identified Fhos, the single Drosophila homolog of the FHOD sub-family of formins, as a primary and versatile mediator of IFM thin-filament organization. Localization of Fhos to the barbed-ends of the arrays, achieved via a novel N-terminal domain, appears to be a critical aspect of its sarcomeric roles. DOI: http://dx.doi.org/10.7554/eLife.16540.001 PMID:27731794

  7. Actin filaments align into hollow comets for rapid VASP-mediated propulsion.

    PubMed

    Plastino, Julie; Olivier, Stéphane; Sykes, Cécile

    2004-10-05

    For cells, the growth of a dense array of branched actin filaments organized by the actin-related proteins 2 and 3 (Arp2/3) complex at the plasma membrane offers an explanation as to how movement is produced, and this arrangement is considered to be optimal for motility. Here, we challenged this assumption by using an in vitro system of polystyrene beads in cell extracts that contained a complex mix of actin polymerization proteins as in vivo. We employed the surface of the bead as a reactor where we mixed two different actin polymerization-activating factors, the Arp2/3 complex and the vasodilator-stimulated phosphoprotein (VASP), to examine their contribution to actin-based movement and filament organization. We varied the coating of the bead surface but left the extracts identical for all assays. We found that the degree of filament alignment in the actin comet tails depended on the surface ratio of VASP to Arp2/3. Alignment of actin filaments parallel to the direction of bead movement in the presence of VASP was accompanied by an abrupt 7-fold increase in velocity that was independent of bead size and by hollowing out of the comets. The actin filament-bundling proteins fimbrin and fascin did not appear to play a role in this transformation. Together with the idea that VASP enhances filament detachment and with the presence of pulling forces at the rear of the bead, a mesoscopic analysis of movement provides a possible explanation for our results.

  8. Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament.

    PubMed

    Masuda, Tadashi

    2013-09-01

    Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the "Driven by Detachment (DbD)" mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.

  9. Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

    PubMed Central

    Cohen, Shenhav; Zhai, Bo; Gygi, Steven P.

    2012-01-01

    During muscle atrophy, myofibrillar proteins are degraded in an ordered process in which MuRF1 catalyzes ubiquitylation of thick filament components (Cohen et al. 2009. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200901052). Here, we show that another ubiquitin ligase, Trim32, ubiquitylates thin filament (actin, tropomyosin, troponins) and Z-band (α-actinin) components and promotes their degradation. Down-regulation of Trim32 during fasting reduced fiber atrophy and the rapid loss of thin filaments. Desmin filaments were proposed to maintain the integrity of thin filaments. Accordingly, we find that the rapid destruction of thin filament proteins upon fasting was accompanied by increased phosphorylation of desmin filaments, which promoted desmin ubiquitylation by Trim32 and degradation. Reducing Trim32 levels prevented the loss of both desmin and thin filament proteins. Furthermore, overexpression of an inhibitor of desmin polymerization induced disassembly of desmin filaments and destruction of thin filament components. Thus, during fasting, desmin phosphorylation increases and enhances Trim32-mediated degradation of the desmin cytoskeleton, which appears to facilitate the breakdown of Z-bands and thin filaments. PMID:22908310

  10. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    PubMed Central

    Carlsson, Anders E

    2010-01-01

    The polymerization of actin via branching at a cell membrane containing nucleation-promoting factors (NPFs) is simulated using a stochastic-growth methodology. The polymerized-actin distribution displays three types of behavior: a) traveling waves, b) moving patches, and c) random fluctuations. Increasing actin concentration causes a transition from patches to waves. The waves and patches move by a treadmilling mechanism which does not require myosin II. The effects of downregulation of key proteins on actin wave behavior are evaluated. PMID:20867207

  11. Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle.

    PubMed

    Gokhin, David S; Tierney, Matthew T; Sui, Zhenhua; Sacco, Alessandra; Fowler, Velia M

    2014-03-01

    Duchenne muscular dystrophy (DMD) induces sarcolemmal mechanical instability and rupture, hyperactivity of intracellular calpains, and proteolytic breakdown of muscle structural proteins. Here we identify the two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, as novel proteolytic targets of m-calpain, with Tmod1 exhibiting ∼10-fold greater sensitivity to calpain-mediated cleavage than Tmod4 in situ. In mdx mice, increased m-calpain levels in dystrophic soleus muscle are associated with loss of Tmod1 from the thin filament pointed ends, resulting in ∼11% increase in thin filament lengths. In mdx/mTR mice, a more severe model of DMD, Tmod1 disappears from the thin filament pointed ends in both tibialis anterior (TA) and soleus muscles, whereas Tmod4 additionally disappears from soleus muscle, resulting in thin filament length increases of ∼10 and ∼12% in TA and soleus muscles, respectively. In both mdx and mdx/mTR mice, both TA and soleus muscles exhibit normal localization of α-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic γ-actin, indicating that m-calpain does not cause wholesale proteolysis of other sarcomeric and actin cytoskeletal proteins in dystrophic skeletal muscle. These results implicate Tmod proteolysis and resultant thin filament length misspecification as novel mechanisms that may contribute to DMD pathology, affecting muscles in a use- and disease severity-dependent manner.

  12. Synthetic chondramide A analogues stabilize filamentous actin and block invasion by Toxoplasma gondii.

    PubMed

    Ma, Christopher I; Diraviyam, Karthikeyan; Maier, Martin E; Sept, David; Sibley, L David

    2013-09-27

    Apicomplexan parasites such as Toxoplasma gondii rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of T. gondii in vitro. Structural modeling of chondramide A (2) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (2b-k) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC₅₀ values that ranged from 0.3 to 1.3 μM. In vitro polymerization assays using highly purified recombinant actin from T. gondii verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites.

  13. Probing the flexibility of tropomyosin and its binding to filamentous actin using molecular dynamics simulations.

    PubMed

    Zheng, Wenjun; Barua, Bipasha; Hitchcock-DeGregori, Sarah E

    2013-10-15

    Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.

  14. Crenactin forms actin-like double helical filaments regulated by arcadin-2

    PubMed Central

    Izoré, Thierry; Kureisaite-Ciziene, Danguole; McLaughlin, Stephen H; Löwe, Jan

    2016-01-01

    The similarity of eukaryotic actin to crenactin, a filament-forming protein from the crenarchaeon Pyrobaculum calidifontis supports the theory of a common origin of Crenarchaea and Eukaryotes. Monomeric structures of crenactin and actin are similar, although their filament architectures were suggested to be different. Here we report that crenactin forms bona fide double helical filaments that show exceptional similarity to eukaryotic F-actin. With cryo-electron microscopy and helical reconstruction we solved the structure of the crenactin filament to 3.8 Å resolution. When forming double filaments, the 'hydrophobic plug' loop in crenactin rearranges. Arcadin-2, also encoded by the arcade gene cluster, binds tightly with its C-terminus to the hydrophobic groove of crenactin. Binding is reminiscent of eukaryotic actin modulators such as cofilin and thymosin β4 and arcadin-2 is a depolymeriser of crenactin filaments. Our work further supports the theory of shared ancestry of Eukaryotes and Crenarchaea. DOI: http://dx.doi.org/10.7554/eLife.21600.001 PMID:27852434

  15. A mechanical model for the motility of actin filaments on myosin

    NASA Astrophysics Data System (ADS)

    Nicolau, Dan V., Jr.; Fulga, Florin; Nicolau, Dan V.

    2004-03-01

    The interaction of actin filaments with myosin is crucial to cell motility, muscular contraction, cell division and other processes. The in vitro motility assay involves the motion of actin filaments on a substrate coated with myosin, and is used extensively to investigate the dynamics of the actomyosin system. Following on from previous work, we propose a new mechanical model of actin motility on myosin, wherein a filament is modeled as a chain of beads connected by harmonic springs. This imposes a limitation on the "stretching" of the filament. The rotation of one bead with respect to its neighbours is also constrained in similar way. We implemented this model and used Monte Carlo simulations to determine whether it can predict the directionality of filament motion. The principal advantages of this model over our previous one are that we have removed the empirically correct but artificial assumption that the filament moves like a "worm" i.e. the head determines the direction of movement and the rest of the filament "follows" the head as well as the inclusion of dependencies on experimental rate constants (and so also on e.g. ATP concentration) via the cross-bridge cycle.

  16. Phototropin-dependent biased relocalization of cp-actin filaments can be induced even when chloroplast movement is inhibited.

    PubMed

    Yamada, Noboru; Suetsugu, Noriyuki; Wada, Masamitsu; Kadota, Akeo

    2011-11-01

    In a recent publication using an actin-visualized line of Arabidopsis (Ichikawa et al. 2011, ref. 11), we reported a detailed analysis with higher time resolution on the dynamics of chloroplast actin filaments (cp-actin filaments) during chloroplast avoidance movement and demonstrated a good correlation between the biased configuration of cp-actin filaments and chloroplast movement. However, we could not conclusively determine whether the reorganization of cp-actin filaments into a biased configuration preceded actual chloroplast movement (and, thus, whether it could be a cause of the movement). In this report, we present clear evidence that the reorganization of cp-actin filaments into a biased distribution is induced even in the absence of the actual movement of chloroplasts. When the cells were treated with 2,3-butanedione monoxime (BDM), a potent inhibitor of myosin ATPase, chloroplast motility was completely suppressed. Nevertheless, the disappearance and biased relocalization of cp-actin filaments toward the side of the prospective movement direction were induced by irradiation with a strong blue light microbeam. The results definitively indicate that the reorganization of cp-actin filaments is not an effect of chloroplast movement; however, it is feasible that the biased localization of cp-actin filaments is an event leading to chloroplast movement.

  17. Rapid severing and motility of chloroplast-actin filaments are required for the chloroplast avoidance response in Arabidopsis.

    PubMed

    Kong, Sam-Geun; Arai, Yoshiyuki; Suetsugu, Noriyuki; Yanagida, Toshio; Wada, Masamitsu

    2013-02-01

    Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light-induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light's intensity and position.

  18. Single-filament kinetic studies provide novel insights into regulation of actin-based motility

    PubMed Central

    Shekhar, Shashank; Carlier, Marie-France

    2016-01-01

    Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles that drive actin remodeling have provided a mechanistic and quantitative understanding of such processes. We specifically consider key barbed-end regulators such as capping protein and formins as illustrative examples. We compare and contrast different kinetic approaches, such as the traditional pyrene-polymerization bulk assays, as well as more recently developed single-filament and single-molecule imaging approaches. Recent development of novel biophysical methods for sensing and applying forces will in future allow us to address the very important relationship between mechanical stimulus and kinetics of actin-based motility. PMID:26715420

  19. Direct actin binding to A- and B-type lamin tails and actin filament bundling by the lamin A tail

    PubMed Central

    Simon, Dan N; Zastrow, Michael S

    2010-01-01

    Nuclear intermediate filament networks formed by A- and B-type lamins are major components of the nucleoskeleton. Lamins have growing links to human physiology and disease including Emery-Dreifuss muscular dystrophy (EDMD), lipodystrophy, cardiomyopathy, neuropathy, cerebellar disorders and segmental accelerated ‘aging’ syndromes. How lamins interact with other nucleoskeletal components, and even the identities of these other components, are open questions. Previous studies suggested lamins might bind actin. We report that the recombinant C-terminal tail domain of human A- and B-type lamins binds directly to purified actin in high-speed pelleting assays. This interaction maps to a conserved Actin Binding site (AB-1) comprising lamin A residues 461–536 in the Ig-fold domain, which are 54% identical in lamin B1. Two EDMD-causing missense mutations (R527P and L530P) in lamin A that are predicted to disrupt the Ig-fold, each reduced F-actin binding by ∼66%, whereas the surface-exposed lipodystrophy-causing R482Q mutation had no significant effect. The lamin A tail was unique among lamins in having a second actin-binding site (AB-2). This second site was mapped to lamin A tail residues 564–608, based on actin-binding results for the lamin C tail and internal deletions in the lamin A tail that cause Hutchinson-Gilford Progeria Syndrome (Δ35, Δ50) or restrictive dermopathy (Δ90). Supporting the presence of two actin-binding sites, recombinant precursor (unmodified) and mature lamin A tails (not C or B1 tails) each bundled F-actin in vitro: furthermore F-actin bundling was reduced 25–40% by the R527P, L530P, Δ35 and Δ50 mutations, and was abolished by Δ90. Unexpectedly, the mature lamin A tail bound F-actin significantly more efficiently than did the prelamin A tail; this suggested unmodified residues 647–664, unique to prelamin A, might auto-inhibit binding to actin (and potentially other partners). These biochemical results suggest direct mechanisms

  20. Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement.

    PubMed

    Ichikawa, Satoshi; Yamada, Noboru; Suetsugu, Noriyuki; Wada, Masamitsu; Kadota, Akeo

    2011-08-01

    The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.

  1. Probing the sliding interactions between bundled actin filaments

    NASA Astrophysics Data System (ADS)

    Ward, Andy; Dogic, Zvonimir

    2012-02-01

    Assemblies of filamentous biopolymers are hierarchical materials in which the properties of the overall assemblage are determined by structure and interactions between constituent particles at all hierarchical levels. For example, the overall bending rigidity of a two bundled filaments greatly depends on the bending rigidity of, and the adhesion strength between individual filaments. However, another property of importance is the ability for the filaments to slide freely against one another. Everyday experience indicates that it is much easier to bend a stack of papers in which individual sheets freely slide past each other than the same stack of papers in which all the sheets are irreversibly glued together. Similarly, in filamentous structures the ability for local re-arrangement is of the utmost importance in determining the properties of the structures observed. We have developed a method to directly measure the frictional interactions between a pair of aligned filaments in a well-defined and controllable configuration. This enables us to systematically investigate the role of adhesion strength, filament orientation, length, and surface structure.

  2. Role of the DNase-I-binding loop in dynamic properties of actin filament.

    PubMed

    Khaitlina, Sofia Yu; Strzelecka-Gołaszewska, Hanna

    2002-01-01

    Effects of proteolytic modifications of the DNase-I-binding loop (residues 39-51) in subdomain 2 of actin on F-actin dynamics were investigated by measuring the rates of the polymer subunit exchange with the monomer pool at steady state and of ATP hydrolysis associated with it, and by determination of relative rate constants for monomer addition to and dissociation from the polymer ends. Cleavage of actin between Gly-42 and Val-43 by protease ECP32 resulted in enhancement of the turnover rate of polymer subunits by an order of magnitude or more, in contrast to less than a threefold increase produced by subtilisin cleavage between Met-47 and Gly-48. Probing the structure of the modified actins by limited digestion with trypsin revealed a correlation between the increased F-actin dynamics and a change in the conformation of subdomain 2, indicating a more open state of the filament subunits relative to intact F-actin. The cleavage with trypsin and steady-state ATPase were cooperatively inhibited by phalloidin, with half-maximal effects at phalloidin to actin molar ratio of 1:8 and full inhibition at a 1:1 ratio. The results support F-actin models in which only the N-terminal segment of loop 39-51 is involved in monomer-monomer contacts, and suggest a possibility of regulation of actin dynamics in the cell through allosteric effects on this segment of the actin polypeptide chain.

  3. The "Le Chatelier's principle"-governed response of actin filaments to osmotic stress.

    PubMed

    Ito, Tadanao; Yamazaki, Masahito

    2006-07-13

    Actin filaments inhibit osmotic stress-driven water flow across a semipermeable membrane in proportion to the filament concentration (Ito, T.; Zaner, K. S.; Stossel, T. P. Biophys. J. 1987, 51, 745). When the filaments are cross-linked by F-actin binding protein, filamin A, this flow is stopped completely (Ito, T.; Suzuki, A.; Stossel, T. P. Biophys. J. 1992, 61, 1301). No conventional theory accurately accounts for these results. Here, this response is analyzed by formulating the entropy of the system under osmotic stress. Results demonstrate that the response of the actin filaments to osmotic stress is governed by the Le Chatelier's principle, which states that an external interaction that disturbs the equilibrium brings about processes in the body that tend to reduce the effects of this interaction. In the present case, disrupting equilibrium by osmotic stress brings about a reaction that decreases the chemical potential of water in the F-actin solution, reducing the effect of the applied osmotic disturbance. This decrease in the chemical potential of the water in the F-actin solution is caused by an increase in the chemical potential of F-actin, which is induced by isothermal absorption of heat by F-actin aided by work done by osmotic stress. As a result, F-actin has an inhibitory effect on the osmotic stress-driven water flow, and can even completely stop the flow when it is cross-linked. This is the first report demonstrating that the Le Chatelier's principle applies to the reaction of biopolymers against equilibrium disturbances such as osmotic stress.

  4. Liquid-like bundles of crosslinked actin filaments contract without motors

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly

    The actin cytoskeleton is a dynamic, structural material that drives cellular-scale deformations during processes such as cell migration and division. Motor proteins are responsible for actively driving many deformations by buckling and translocating actin filaments. However, there is evidence that deformations, such as the constriction of the actin bundle that drives the separation of cells during division, can occur without motors, mediated instead by crosslinker proteins. How might crosslinkers, independent of motors, drive contraction of a bundle? Using a model system of purified proteins, we show that crosslinkers, analogous to molecular cohesion, create an effective surface tension that induces bundle contraction. Crosslinked short actin filaments form micron-sized spindle-shaped bundles. Similar to tactoid granules found at the isotropic-nematic phase transition in liquid crystals, these bundles coarsen and coalesce like liquid droplets. In contrast, crosslinked long filaments coarsen into a steady state of bundles that are frozen in a solid-like network. Near the liquid-solid boundary, filaments of intermediate length initially form bundles that spontaneously contract into tactoid droplets. Our results, that crosslinked actin bundles are liquid-like with an effective surface tension, provide evidence for a mechanism of motor-independent contractility in biological materials.

  5. Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation

    PubMed Central

    Jiang, Shimin; Narita, Akihiro; Popp, David; Ghoshdastider, Umesh; Lee, Lin Jie; Srinivasan, Ramanujam; Balasubramanian, Mohan K.; Oda, Toshiro; Koh, Fujiet; Larsson, Mårten; Robinson, Robert C.

    2016-01-01

    Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule. PMID:26873105

  6. Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape

    PubMed Central

    1993-01-01

    Microtubules are long, proteinaceous filaments that perform structural functions in eukaryotic cells by defining cellular shape and serving as tracks for intracellular motor proteins. We report the first accurate measurements of the flexural rigidity of microtubules. By analyzing the thermally driven fluctuations in their shape, we estimated the mean flexural rigidity of taxol-stabilized microtubules to be 2.2 x 10(-23) Nm2 (with 6.4% uncertainty) for seven unlabeled microtubules and 2.1 x 10(-23) Nm2 (with 4.7% uncertainty) for eight rhodamine-labeled microtubules. These values are similar to earlier, less precise estimates of microtubule bending stiffness obtained by modeling flagellar motion. A similar analysis on seven rhodamine-phalloidin- labeled actin filaments gave a flexural rigidity of 7.3 x 10(-26) Nm2 (with 6% uncertainty), consistent with previously reported results. The flexural rigidity of these microtubules corresponds to a persistence length of 5,200 microns showing that a microtubule is rigid over cellular dimensions. By contrast, the persistence length of an actin filament is only approximately 17.7 microns, perhaps explaining why actin filaments within cells are usually cross-linked into bundles. The greater flexural rigidity of a microtubule compared to an actin filament mainly derives from the former's larger cross-section. If tubulin were homogeneous and isotropic, then the microtubule's Young's modulus would be approximately 1.2 GPa, similar to Plexiglas and rigid plastics. Microtubules are expected to be almost inextensible: the compliance of cells is due primarily to filament bending or sliding between filaments rather than the stretching of the filaments themselves. PMID:8432732

  7. Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments.

    PubMed

    Beach, Jordan R; Bruun, Kyle S; Shao, Lin; Li, Dong; Swider, Zac; Remmert, Kirsten; Zhang, Yingfan; Conti, Mary A; Adelstein, Robert S; Rusan, Nasser M; Betzig, Eric; Hammer, John A

    2017-02-01

    The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

  8. Effects of solution crowding on actin polymerization reveal the energetic basis for nucleotide-dependent filament stability

    PubMed Central

    Frederick, Kendra B.; Sept, David; De La Cruz, Enrique M.

    2008-01-01

    Actin polymerization is a fundamental cellular process involved in cell structure maintenance, force generation, and motility. Phosphate release from filament subunits following ATP hydrolysis destabilizes the filament lattice and increases the critical concentration (Cc) for assembly. The structural differences between ATP- and ADP-actin are still debated, as well as the energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded intracellular conditions. Here, we investigate the effect of crowding agents on ATP- and ADP-actin polymerization, and find that ATP-actin polymerization is largely unaffected by solution crowding, while crowding agents lower the Cc of ADP-actin in a concentration-dependent manner. The stabilities of ATP- and ADP-actin filaments are comparable in the presence of physiological amounts (~30% w/v) and types (sorbitol) of low molecular weight crowding agents. Crowding agents act to stabilize ADP-F-actin by slowing subunit dissociation. These observations suggest that nucleotide hydrolysis and phosphate release per se do not introduce intrinsic differences in the in vivo filament stability. Rather, the preferential disassembly of ADP-actin filaments in cells is driven through interactions with regulatory proteins. Interpretation of the experimental data according to osmotic stress theory implicates water as an allosteric regulator of actin activity and hydration as the molecular basis for nucleotide-dependent filament stability. PMID:18374941

  9. Filament compliance influences cooperative activation of thin filaments and the dynamics of force production in skeletal muscle.

    PubMed

    Tanner, Bertrand C W; Daniel, Thomas L; Regnier, Michael

    2012-01-01

    Striated muscle contraction is a highly cooperative process initiated by Ca²⁺ binding to the troponin complex, which leads to tropomyosin movement and myosin cross-bridge (XB) formation along thin filaments. Experimental and computational studies suggest skeletal muscle fiber activation is greatly augmented by cooperative interactions between neighboring thin filament regulatory units (RU-RU cooperativity; 1 RU = 7 actin monomers+1 troponin complex+1 tropomyosin molecule). XB binding can also amplify thin filament activation through interactions with RUs (XB-RU cooperativity). Because these interactions occur with a temporal order, they can be considered kinetic forms of cooperativity. Our previous spatially-explicit models illustrated that mechanical forms of cooperativity also exist, arising from XB-induced XB binding (XB-XB cooperativity). These mechanical and kinetic forms of cooperativity are likely coordinated during muscle contraction, but the relative contribution from each of these mechanisms is difficult to separate experimentally. To investigate these contributions we built a multi-filament model of the half sarcomere, allowing RU activation kinetics to vary with the state of neighboring RUs or XBs. Simulations suggest Ca²⁺ binding to troponin activates a thin filament distance spanning 9 to 11 actins and coupled RU-RU interactions dominate the cooperative force response in skeletal muscle, consistent with measurements from rabbit psoas fibers. XB binding was critical for stabilizing thin filament activation, particularly at submaximal Ca²⁺ levels, even though XB-RU cooperativity amplified force less than RU-RU cooperativity. Similar to previous studies, XB-XB cooperativity scaled inversely with lattice stiffness, leading to slower rates of force development as stiffness decreased. Including RU-RU and XB-RU cooperativity in this model resulted in the novel prediction that the force-[Ca²⁺] relationship can vary due to filament and XB

  10. Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments[W

    PubMed Central

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca2+, in vitro. Analysis of a mutant that bears a point mutation at the Ca2+ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca2+ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth. PMID:24424096

  11. Live Cell Imaging of Actin Dynamics in the Filamentous Fungus Aspergillus nidulans.

    PubMed

    Schultzhaus, Zachary; Quintanilla, Laura; Hilton, Angelyn; Shaw, Brian D

    2016-04-01

    Hyphal cells of filamentous fungi grow at their tips in a method analogous to pollen tube and root hair elongation. This process, generally referred to as tip growth, requires precise regulation of the actin cytoskeleton, and characterizing the various actin structures in these cell types is currently an active area of research. Here, the actin marker Lifeact was used to document actin dynamics in the filamentous fungus Aspergillus nidulans. Contractile double rings were observed at septa, and annular clusters of puncta were seen subtending growing hyphal tips, corresponding to the well-characterized subapical endocytic collar. However, Lifeact also revealed two additional structures. One, an apical array, was dynamic on the face opposite the tip, while a subapical web was dynamic on the apical face and was located several microns behind the growth site. Each was observed turning into the other over time, implying that they could represent different localizations of the same structure, although hyphae with a subapical web grew faster than those exhibiting an apical array. The subapical web has not been documented in any filamentous fungus to date, and is separate from the networks of F-actin seen in other tip-growing organisms surrounding septa or stationary along the plasmalemma.

  12. Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites.

    PubMed

    Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A; Strack, Stefan; Higgs, Henry N

    2015-11-26

    While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.

  13. Direct interaction of actin filaments with F-BAR protein pacsin2

    PubMed Central

    Kostan, Julius; Salzer, Ulrich; Orlova, Albina; Törö, Imre; Hodnik, Vesna; Senju, Yosuke; Zou, Juan; Schreiner, Claudia; Steiner, Julia; Meriläinen, Jari; Nikki, Marko; Virtanen, Ismo; Carugo, Oliviero; Rappsilber, Juri; Lappalainen, Pekka; Lehto, Veli-Pekka; Anderluh, Gregor; Egelman, Edward H; Djinović-Carugo, Kristina

    2014-01-01

    Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes. PMID:25216944

  14. A variational approach to the growth dynamics of pre-stressed actin filament networks

    NASA Astrophysics Data System (ADS)

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-01

    In order to model the growth dynamics of elastic bodies with residual stresses a thermodynamically consistent approach is needed such that the cross-coupling between growth and mechanics can be correctly described. In the present work we apply a variational principle to the formulation of the interfacial growth dynamics of dendritic actin filament networks growing from biomimetic beads, an experimentally well studied system, where the buildup of residual stresses governs the network growth. We first introduce the material model for the network via a strain energy density for an isotropic weakly nonlinear elastic material and then derive consistently from this model the dynamic equations for the interfaces, i.e. for a polymerizing internal interface in contact with the bead and a depolymerizing external interface directed towards the solvent. We show that (i) this approach automatically preserves thermodynamic symmetry-properties, which is not the case for the often cited ‘rubber-band-model’ (Sekimoto et al 2004 Eur. Phys. J. E 13 247-59, Plastino et al 2004 Eur. Biophys. J. 33 310-20) and (ii) leads to a robust morphological instability of the treadmilling network interfaces. The nature of the instability depends on the interplay of the two dynamic interfaces. Depending on the biochemical conditions the network envelope evolves into a comet-like shape (i.e. the actin envelope thins out at one side and thickens on the opposite side of the bead) via a varicose instability or it breaks the symmetry via higher order zigzag modes. We conclude that morphological instabilities due to mechano-chemical coupling mechanisms and the presences of mechancial pre-stresses can play a major role in locally organizing the cytoskeleton of living cells.

  15. Extracellular Inhibitors, Repellents, and Semaphorin/Plexin/MICAL-mediated Actin Filament Disassembly

    PubMed Central

    Hung, Ruei-Jiun; Terman, Jonathan R.

    2011-01-01

    Multiple extracellular signals have been identified that regulate actin dynamics within motile cells, but how these instructive cues present on the cell surface exert their precise effects on the internal actin cytoskeleton is still poorly understood. One particularly interesting class of these cues is a group of extracellular proteins that negatively alter the movement of cells and their processes. Over the years, these types of events have been described using a variety of terms and herein we provide an overview of inhibitory/repulsive cellular phenomena and highlight the largest known protein family of repulsive extracellular cues, the Semaphorins. Specifically, the Semaphorins (Semas) utilize Plexin cell-surface receptors to dramatically collapse the actin cytoskeleton and we summarize what is known of the direct molecular and biochemical mechanisms of Sema-triggered actin filament (F-actin) disassembly. We also discuss new observations from our lab that reveal that the multi-domain oxidoreductase (Redox) enzyme MICAL, an important mediator of Sema/Plexin repulsion, is a novel F-actin disassembly factor. Our results indicate that MICAL triggers Sema/Plexin-mediated reorganization of the F-actin cytoskeleton and suggest a role for specific Redox signaling events in regulating actin dynamics. PMID:21800438

  16. Real-Time Measurements of Actin Filament Polymerization by Total Internal Reflection Fluorescence Microscopy

    PubMed Central

    Kuhn, Jeffrey R.; Pollard, Thomas D.

    2005-01-01

    Understanding the mechanism of actin polymerization and its regulation by associated proteins requires an assay to monitor polymerization dynamics and filament topology simultaneously. The only assay meeting these criteria is total internal reflection fluorescence microscopy (Amann and Pollard, 2001; Fujiwara et al., 2002). The fluorescence signal is fourfold stronger with actin labeled on Cys-374 with Oregon green rather than rhodamine. To distinguish growth at barbed and pointed ends we used image drift correction and maximum intensity projections to reveal points where single N-ethylmaleimide inactivated myosins attach filaments to the glass coverslip. We estimated association rates at high actin concentrations and dissociation rates near and below the critical actin concentration. At the barbed end, the association rate constant for Mg-ATP-actin is 7.4 μM−1 s−1 and the dissociation rate constant is 0.89 s−1. At the pointed end the association and dissociation rate constants are 0.56 μM−1 s−1 and 0.19 s−1. When vitamin D binding protein sequesters all free monomers, ADP-actin dissociates from barbed ends at 1.4 s−1 and from pointed ends at 0.16 s−1 regardless of buffer nucleotide. PMID:15556992

  17. Control of actin filament dynamics at barbed ends by WH2 domains: from capping to permissive and processive assembly.

    PubMed

    Carlier, Marie-France; Pernier, Julien; Avvaru, Balendu Sankara

    2013-10-01

    WH2 domains are multifunctional regulators of actin assembly that can either sequester G-actin or allow polarized barbed end growth. They all bind similarly to a hydrophobic pocket at the barbed face of actin. Depending on their electrostatic environment, WH2 domains can nucleate actin assembly by facilitating the formation of prenuclei dimers along the canonical spontaneous assembly pathway. They also modulate filament barbed end dynamics in a versatile fashion, acting either as barbed end cappers or assisting barbed end growth like profilin or uncapping barbed ends and potentially mediating processive elongation like formins when they are dimerized. Tandem repeats of WH2 domains can sever filaments and either remain bound to created barbed ends like gelsolin, or strip off an ADP-actin subunit from the severed polymer end, depending on their relative affinity for terminal ADP-F-actin or ADP-G-actin. In summary, WH2 domains recapitulate all known elementary regulatory functions so far found in individual actin-binding proteins. By combining different discrete sets of these multifunctional properties, they acquire specific functions in various actin-based processes, and participate in activities as diverse as filament branching, filopodia extension, or actin remodeling in ciliogenesis and asymmetric meiotic division. They also integrate these functions with other actin-binding motifs present either in the same protein or in a complex with another protein, expanding the range of complexity in actin regulation. The details of their molecular mechanisms and the underlying structural basis provide exciting avenues in actin research.

  18. The Role of Formin Tails in Actin Nucleation, Processive Elongation, and Filament Bundling*

    PubMed Central

    Vizcarra, Christina L.; Bor, Batbileg; Quinlan, Margot E.

    2014-01-01

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements. PMID:25246531

  19. Direct observation of motion of single F-actin filaments in the presence of myosin

    NASA Astrophysics Data System (ADS)

    Yanagida, Toshio; Nakase, Michiyuki; Nishiyama, Katsumi; Oosawa, Fumio

    1984-01-01

    Actin is found in almost all kinds of non-muscle cells where it is thought to have an important role in cell motility. A proper understanding of that role will only be possible when reliable in vitro systems are available for investigating the interaction of cellular actin and myosin. A start has been made on several systems1-4, most recently by Sheetz and Spudich who demonstrated unidirectional movement of HMM-coated beads along F-actin cables on arrays of chloroplasts exposed by dissection of a Nitella cell5. As an alternative approach, we report here the direct observation by fluorescence microscopy of the movements of single F-actin filaments interacting with soluble myosin fragments energized by Mg2+-ATP.

  20. A coarse-grained model to study calcium activation of the cardiac thin filament

    NASA Astrophysics Data System (ADS)

    Zhang, Jing; Schwartz, Steven

    2015-03-01

    Familial hypertrophic cardiomyopathy (FHC) is one of the most common heart disease caused by genetic mutations. Cardiac muscle contraction and relaxation involve regulation of crossbridge binding to the cardiac thin filament, which regulates actomyosin interactions through calcium-dependent alterations in the dynamics of cardiac troponin (cTn) and tropomyosin (Tm). An atomistic model of cTn complex interacting with Tm has been studied by our group. A more realistic model requires the inclusion of the dynamics of actin filament, which is almost 6 times larger than cTn and Tm in terms of atom numbers, and extensive sampling of the model becomes very resource-demanding. By using physics-based protein united-residue force field, we introduce a coarse-grained model to study the calcium activation of the thin filament resulting from cTn's allosteric regulation of Tm dynamics on actin. The time scale is much longer than that of all-atom molecular dynamics simulation because of the reduction of the degrees of freedom. The coarse-grained model is a good template for studying cardiac thin filament mutations that cause FHC, and reduces the cost of computational resources.

  1. Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

    PubMed Central

    Yuen, Michaela; Sandaradura, Sarah A.; Dowling, James J.; Kostyukova, Alla S.; Moroz, Natalia; Quinlan, Kate G.; Lehtokari, Vilma-Lotta; Ravenscroft, Gianina; Todd, Emily J.; Ceyhan-Birsoy, Ozge; Gokhin, David S.; Maluenda, Jérome; Lek, Monkol; Nolent, Flora; Pappas, Christopher T.; Novak, Stefanie M.; D’Amico, Adele; Malfatti, Edoardo; Thomas, Brett P.; Gabriel, Stacey B.; Gupta, Namrata; Daly, Mark J.; Ilkovski, Biljana; Houweling, Peter J.; Davidson, Ann E.; Swanson, Lindsay C.; Brownstein, Catherine A.; Gupta, Vandana A.; Medne, Livija; Shannon, Patrick; Martin, Nicole; Bick, David P.; Flisberg, Anders; Holmberg, Eva; Van den Bergh, Peter; Lapunzina, Pablo; Waddell, Leigh B.; Sloboda, Darcée D.; Bertini, Enrico; Chitayat, David; Telfer, William R.; Laquerrière, Annie; Gregorio, Carol C.; Ottenheijm, Coen A.C.; Bönnemann, Carsten G.; Pelin, Katarina; Beggs, Alan H.; Hayashi, Yukiko K.; Romero, Norma B.; Laing, Nigel G.; Nishino, Ichizo; Wallgren-Pettersson, Carina; Melki, Judith; Fowler, Velia M.; MacArthur, Daniel G.; North, Kathryn N.; Clarke, Nigel F.

    2014-01-01

    Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle. PMID:25250574

  2. Tropomyosin-dependent filament formation by a polymerization-defective mutant yeast actin (V266G,L267G).

    PubMed

    Wen, K K; Kuang, B; Rubenstein, P A

    2000-12-22

    A major function of tropomyosin (TPM) in nonmuscle cells may be stabilization of F-actin by binding longitudinally along the actin filament axis. However, no clear evidence exists in vitro that TPM can significantly affect the critical concentration of actin. We previously made a polymerization-defective mutant actin, GG (V266G, L267G). This actin will not polymerize alone at 25 degrees C but will in the presence of phalloidin or beryllium fluoride. With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive. We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25 degrees C with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac tropomyosin), indicating the importance of the ends in polymerization rescue. In the presence of TPM, the apparent critical concentration of actin is 5.5 microm, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 microm). Non N-acetylated TPMs did not rescue GG-actin polymerization. The TPMs did not prevent cold-induced depolymerization of GG F-actin. TPM-dependent GG-actin polymerization did not occur at temperatures below 20 degrees C. Polymerization rescue may depend initially on the capture of unstable GG-F-actin oligomers by the TPM, resulting in the strengthening of actin monomer-monomer contacts along the filament axis.

  3. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments

    PubMed Central

    Heaslip, Aoife T.; Nelson, Shane R.; Warshaw, David M.

    2016-01-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite’s intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein–tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

  4. Caenorhabditis elegans Kettin, a Large Immunoglobulin-like Repeat Protein, Binds to Filamentous Actin and Provides Mechanical Stability to the Contractile Apparatuses in Body Wall Muscle

    PubMed Central

    Ono, Kanako; Yu, Robinson; Mohri, Kurato

    2006-01-01

    Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with α-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction. PMID:16597697

  5. Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

    PubMed Central

    1995-01-01

    The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion. PMID:7490299

  6. Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

    PubMed

    Bharat, Tanmay A M; Murshudov, Garib N; Sachse, Carsten; Löwe, Jan

    2015-07-02

    Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

  7. Multi-photon Imaging of Actin Filament Formation and Mitochondrial Energetics of Human ACBT Gliomas

    PubMed Central

    Hwang, Yu-Jer; Kolettis, Nomiki; Yang, Miso; Gillard, Elizabeth R.; Sanchez, Edgar; Sun, Chung-ho; Tromberg, Bruce J.; Krasieva, Tatiana B.; Lyubovitsky, Julia G.

    2011-01-01

    We studied the three-dimensional (3D) distribution of actin filaments and mitochondria in relation to ACBT glioblastoma cells migration. We embedded the cells in the spheroid form within collagen hydrogels and imaged them by in-situ multi-photon microscopy (MPM). The static 3D overlay of the distribution of actin filaments and mitochondria provided a greater understanding of cell-to-cell and cell-to-substrate interactions and morphology. While imaging mitochondria to obtain ratiometric redox index based on cellular fluorescence from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) we observed differential sensitivity of the migrating ACBT glioblastoma cells to femtosecond laser irradiation employed in MPM. We imaged actin-GFP fluorescence in live ACBT glioma cells and for the first time observed dynamic modulation of the pools of actin during migration in 3D. The MPM imaging, which probes cells directly within the 3D cancer models, could potentially aid in working out a link between the functional performance of mitochondria, actin distribution and cancer invasiveness. PMID:21143483

  8. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins.

    PubMed

    Blanchoin, L; Amann, K J; Higgs, H N; Marchand, J B; Kaiser, D A; Pollard, T D

    2000-04-27

    Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.

  9. Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy

    PubMed Central

    Ngo, Kien Xuan; Kodera, Noriyuki; Katayama, Eisaku; Ando, Toshio; Uyeda, Taro QP

    2015-01-01

    High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ∼25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries. DOI: http://dx.doi.org/10.7554/eLife.04806.001 PMID:25642645

  10. ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin.

    PubMed

    Nomura, Kazumi; Ono, Shoichiro

    2013-07-15

    CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP- or ADP-actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer-filament equilibrium.

  11. Asymmetric Myosin Binding to the Thin Filament as Revealed by a Fluorescent Nanocircuit

    PubMed Central

    Coffee Castro-Zena, Pilar G.; Root, Douglas D.

    2013-01-01

    The interplay between myosin, actin, and striated muscle regulatory proteins involves complex cooperative interactions that propagate along the thin filament. A repeating unit of the tropomyosin dimer, troponin heterotrimer, and the actin protofilament heptamer is sometimes assumed to be able to bind myosin at any of its seven actins when activated even though the regulatory proteins are asymmetrically positioned along this repeating unit. Analysis of the impact of this asymmetry on actin and myosin interactions by sensitized emission luminescence resonance energy transfer spectroscopy and a unique fluorescent nanocircuit design reveals that the troponin affects the structure and function of myosin heads bound nearby in a different manner than myosin heads bound further away from the troponin. To test this hypothesis, a fluorescent nanocircuit reported the position of the myosin lever arm only when the myosin was bound adjacent to the troponin, or in controls, only when the myosin was bound distant from the troponin. Confirming the hypothesis, the myosin lever arm is predominantly in the prepowerstroke orientation when bound near troponin, but is predominantly in the postpowerstroke orientation when bound distant from troponin. These data are consistent with the hypothesis that troponin is responsible for the formation of myosin binding target zones along the thin filament. PMID:23274408

  12. Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

    PubMed Central

    Kronlage, Cornelius; Schäfer-Herte, Marco; Böning, Daniel; Oberleithner, Hans; Fels, Johannes

    2015-01-01

    Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane. PMID:26287621

  13. Stabilizing the Central Part of Tropomyosin Increases the Bending Stiffness of the Thin Filament

    PubMed Central

    Nabiev, Salavat R.; Ovsyannikov, Denis A.; Kopylova, Galina V.; Shchepkin, Daniil V.; Matyushenko, Alexander M.; Koubassova, Natalia A.; Levitsky, Dmitrii I.; Tsaturyan, Andrey K.; Bershitsky, Sergey Y.

    2015-01-01

    A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness. PMID:26200873

  14. Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

    PubMed Central

    Yehl, Jenna; Kudryashova, Elena; Reisler, Emil; Kudryashov, Dmitri; Polenova, Tatyana

    2017-01-01

    Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin’s involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin. PMID:28303963

  15. Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

    2013-03-01

    The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

  16. MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots

    PubMed Central

    Jacques, Eveline; Lewandowski, Michal; Buytaert, Jan; Fierens, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

    2013-01-01

    The plant cytoskeleton plays a crucial role in the cells’ growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton. PMID:23656865

  17. Impact of actin filament stabilization on adult hippocampal and olfactory bulb neurogenesis.

    PubMed

    Kronenberg, Golo; Gertz, Karen; Baldinger, Tina; Kirste, Imke; Eckart, Sarah; Yildirim, Ferah; Ji, Shengbo; Heuser, Isabella; Schröck, Helmut; Hörtnagl, Heide; Sohr, Reinhard; Djoufack, Pierre Chryso; Jüttner, René; Glass, Rainer; Przesdzing, Ingo; Kumar, Jitender; Freyer, Dorette; Hellweg, Rainer; Kettenmann, Helmut; Fink, Klaus Benno; Endres, Matthias

    2010-03-03

    Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca(2+)-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here, we used gelsolin-deficient (Gsn(-/-)) mice as a model system for actin filament stabilization. In Gsn(-/-) mice, emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro, gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly, hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca(2+)](i) increases and exocytotic neurotransmitter release were enhanced in Gsn(-/-) synaptosomes. Importantly, treatment of Gsn(-/-) synaptosomes with mycotoxin cytochalasin D, which, like gelsolin, produces actin disassembly, decreased enhanced Ca(2+) influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly, depolarization-induced glutamate release from Gsn(-/-) brain slices was increased. Furthermore, increased hippocampal neurogenesis in Gsn(-/-) mice was associated with a special microenvironment characterized by enhanced density of perfused vessels, increased regional cerebral blood flow, and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together, reduced filamentous actin turnover in presynaptic terminals causes increased Ca(2+) influx and, subsequently, elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn(-/-) hippocampus is associated with a special vascular niche for neurogenesis.

  18. High expression of Lifeact in Arabidopsis thaliana reduces dynamic reorganization of actin filaments but does not affect plant development.

    PubMed

    van der Honing, Hannie S; van Bezouwen, Laura S; Emons, Anne Mie C; Ketelaar, Tijs

    2011-10-01

    Lifeact is a novel probe that labels actin filaments in a wide range of organisms. We compared the localization and reorganization of Lifeact:Venus-labeled actin filaments in Arabidopsis root hairs and root epidermal cells of lines that express different levels of Lifeact: Venus with that of actin filaments labeled with GFP:FABD2, a commonly used probe in plants. Unlike GFP:FABD2, Lifeact:Venus labeled the highly dynamic fine F-actin in the subapical region of tip-growing root hairs. Lifeact:Venus expression at varying levels was not observed to affect plant development. However, at expression levels comparable to those of GFP:FABD2 in a well-characterized marker line, Lifeact:Venus reduced reorganization rates of bundles of actin filaments in root epidermal cells. Reorganization rates of cytoplasmic strands, which reflect the reorganization of the actin cytoskeleton, were also reduced in these lines. Moreover, in the same line, Lifeact:Venus-decorated actin filaments were more resistant to depolymerization by latrunculin B than those in an equivalent GFP:FABD2-expressing line. In lines where Lifeact: Venus is expressed at lower levels, these effects are less prominent or even absent. We conclude that Lifeact: Venus reduces remodeling of the actin cytoskeleton in Arabidopsis in a concentration-dependent manner. Since this reduction occurs at expression levels that do not cause defects in plant development, selection of normally growing plants is not sufficient to determine optimal Lifeact expression levels. When correct expression levels of Lifeact have been determined, it is a valuable probe that labels dynamic populations of actin filaments such as fine F-actin, better than FABD2 does.

  19. Electric field modulation of the motility of actin filaments on myosin-functionalised surfaces

    NASA Astrophysics Data System (ADS)

    Ramsey, L. C.; Aveyard, J.; van Zalinge, H.; Persson, M.; Mânsson, A.; Nicolau, D. V.

    2013-02-01

    We investigated the difference in electrically guided acto-myosin motility on two surfaces. Rabbit skeletal muscle heavy meromyosin (HMM) was absorbed onto surfaces coated with Nitrocellulose (NC) and Poly(butyl methacrylate) (PBMA). A modified in vitro motility assay with sealed chambers for the insertion of electrodes allowed an electrical field to be applied across the flow cell. On all surfaces a small increase in velocity and general guidance of the actin filaments towards the positive electrode is seen at field strengths in the range of ~3000 - 4000Vm-1. A large increase in velocity was observed at ~5000Vm-1 and a significant change in the velocity of the actin filaments present in field strengths higher than this. NC supported the highest percentage of motile filaments and at a field of 8000Vm-1 reached ~66%. PBMA however supported the least percentage of motile filaments and irregular motility was observed even at higher fields where guidance was expected to be strong. The change in velocity in the range of fields tested varied significantly on the surfaces with NC displaying a 46% increase from 0 to 8000Vm-1 whereas on PBMA this value was just 37%.

  20. Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells.

    PubMed

    Laine, R O; Phaneuf, K L; Cunningham, C C; Kwiatkowski, D; Azuma, T; Southwick, F S

    1998-08-01

    The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed

  1. Visualization and force measurement of branching by Arp2/3 complex and N-WASP in actin filament.

    PubMed

    Fujiwara, Ikuko; Suetsugu, Shiro; Uemura, Sotaro; Takenawa, Tadaomi; Ishiwata, Shin'ichi

    2002-05-24

    To determine whether the Arp2/3 complex activated by N-WASP (VCA) branches actin filaments at the side (side branching), or at the barbed (B-)end (end branching) of the mother filaments, we have directly observed the branching process of actin filaments and examined single-molecule unbinding under optical microscope. We found that side branching was predominant, though not exclusive. At the initial stage of polymerization, the branching at the B-end occurred and subsequently the side branching started to occur. In either type of branching, the mother and daughter filaments elongated at nearly the same rate (growing type). Independently of the stage of polymerization, branching due to the direct coupling of filaments with an acute angle to the mother filaments (a coupling type) occurred. Phalloidin suppressed the growing type of branching but not the coupling type, implying that actin monomers are required for the former but not the latter. We found, by single molecule measurements using optical tweezers, that the Arp2/3 complex attaches to the side of actin filaments and the N-WASP appears to detach from the actin-Arp2/3 complex at 6-7 pN.

  2. Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

    PubMed Central

    Periz, Javier; Whitelaw, Jamie; Harding, Clare; Gras, Simon; Del Rosario Minina, Mario Igor; Latorre-Barragan, Fernanda; Lemgruber, Leandro; Reimer, Madita Alice; Insall, Robert; Heaslip, Aoife; Meissner, Markus

    2017-01-01

    Apicomplexan actin is important during the parasite's life cycle. Its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. It has not been possible to study actin in vivo and so its physiological roles have remained obscure, leading to models distinct from conventional actin behaviour. Here a modified version of the commercially available actin-chromobody was tested as a novel tool for visualising F-actin dynamics in Toxoplasma gondii. Cb labels filamentous actin structures within the parasite cytosol and labels an extensive F-actin network that connects parasites within the parasitophorous vacuole and allows vesicles to be exchanged between parasites. In the absence of actin, parasites lack a residual body and inter-parasite connections and grow in an asynchronous and disorganized manner. Collectively, these data identify new roles for actin in the intracellular phase of the parasites lytic cycle and provide a robust new tool for imaging parasitic F-actin dynamics. DOI: http://dx.doi.org/10.7554/eLife.24119.001 PMID:28322189

  3. Mesoscopic model for filament orientation in growing actin networks: the role of obstacle geometry

    NASA Astrophysics Data System (ADS)

    Weichsel, Julian; Schwarz, Ulrich S.

    2013-03-01

    Propulsion by growing actin networks is a universal mechanism used in many different biological systems, ranging from the sheet-like lamellipodium of crawling animal cells to the actin comet tails induced by certain bacteria and viruses in order to move within their host cells. Although the core molecular machinery for actin network growth is well preserved in all of these cases, the geometry of the propelled obstacle varies considerably. During recent years, filament orientation distribution has emerged as an important observable characterizing the structure and dynamical state of the growing network. Here we derive several continuum equations for the orientation distribution of filaments growing behind stiff obstacles of various shapes and validate the predicted steady state orientation patterns by stochastic computer simulations based on discrete filaments. We use an ordinary differential equation approach to demonstrate that for flat obstacles of finite size, two fundamentally different orientation patterns peaked at either ±35° or +70°/0°/ - 70° exhibit mutually exclusive stability, in agreement with earlier results for flat obstacles of very large lateral extension. We calculate and validate phase diagrams as a function of model parameters and show how this approach can be extended to obstacles with piecewise straight contours. For curved obstacles, we arrive at a partial differential equation in the continuum limit, which again is in good agreement with the computer simulations. In all cases, we can identify the same two fundamentally different orientation patterns, but only within an appropriate reference frame, which is adjusted to the local orientation of the obstacle contour. Our results suggest that two fundamentally different network architectures compete with each other in growing actin networks, irrespective of obstacle geometry, and clarify how simulated and electron tomography data have to be analyzed for non-flat obstacle geometries.

  4. Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging

    PubMed Central

    Smith, Benjamin A.; Daugherty-Clarke, Karen; Goode, Bruce L.; Gelles, Jeff

    2013-01-01

    Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ∼1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors. PMID:23292935

  5. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

    PubMed Central

    Brzoska, Anthony J.; Jensen, Slade O.; Barton, Deborah A.; Davies, Danielle S.; Overall, Robyn L.; Skurray, Ronald A.; Firth, Neville

    2016-01-01

    Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments. PMID:27310470

  6. Abundance of actin filaments in the preprophase band and mitotic spindle of brick1 Zea mays mutant.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Tzioutziou, Nickoleta A

    2009-07-01

    The preprophase band and mitotic spindle of dividing protodermal cells of wild-type Zea mays leaves include few actin filaments. Surprisingly, abundant actin filaments were observed in the above arrays, in dividing protodermal cells in the leaves of the brick1 mutant. The same abundance was observed in the spindle of Taxol-treated brick1 mitotic protodermal cells. Apart from the above difference, the relevant arrays displayed normal microtubule organization in both wild type and mutant cells, as far as can be discerned by immunofluorescence microscopy. Accordingly, the abundance of actin filaments in the preprophase band and spindle of brick1 mitotic cells seems not to influence the structure of the above arrays and might be a non-functional "side-effect" of defective F-actin organization in this mutant.

  7. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

    PubMed

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-10-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation.

  8. Myosin-Va binds to and mechanochemically couples microtubules to actin filaments.

    PubMed

    Cao, Tracy T; Chang, Wakam; Masters, Sarah E; Mooseker, Mark S

    2004-01-01

    Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.

  9. Closed membrane shapes with attached BAR domains subject to external force of actin filaments.

    PubMed

    Mesarec, Luka; Góźdź, Wojciech; Iglič, Veronika Kralj; Kralj, Samo; Iglič, Aleš

    2016-05-01

    Membrane deformations induced by attached BAR superfamily domains could trigger or facilitate the growth of plasma membrane protrusions. The BAR domain family consists of BAR, F-BAR and I-BAR domains, each enforcing a different local curvature when attached to the membrane surface. Our theoretical study mainly focuses on the role of I-BAR in the membrane tubular deformations generated or stabilised by actin filaments. The influence of the area density of membrane attached BAR domains and their intrinsic curvature on the closed membrane shapes (vesicles) was investigated numerically. We derived an analytical approximative expression for the critical relative area density of BARs at which the membrane tubular protrusions on vesicles are most prominent. We have shown that the BARs with a higher intrinsic curvature induce thinner and longer cylindrical protrusions. The average orientation of the membrane attached BARs is altered when the vesicle shape is subjected to external force of growing actin rod-like structure inside a vesicle. The average orientation angle of membrane attached BARs may indicate whether the actin filaments are just stabilising the protrusion or generating it by stretching the vesicle.

  10. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments

    PubMed Central

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C.; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-01-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H+-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation. PMID:26272553

  11. Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

    PubMed

    Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R

    2015-07-06

    Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

  12. The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor.

    PubMed

    Ono, Shoichiro

    2013-08-01

    Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in actin filament dynamics. CAP enhances the recharging of actin monomers with ATP antagonistically to ADF/cofilin, and also promotes the severing of actin filaments in cooperation with ADF/cofilin. Self-oligomerization and binding to other proteins regulate activities and localization of CAP. CAP has crucial roles in cell signaling, development, vesicle trafficking, cell migration and muscle sarcomere assembly. This Commentary discusses the recent advances in our understanding of the functions of CAP and its implications as an important regulator of actin cytoskeletal dynamics, which are involved in various cellular activities.

  13. Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond.

    PubMed

    Zhou, Guo-Lei; Zhang, Haitao; Field, Jeffrey

    2014-01-01

    Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.

  14. Gas7b (growth arrest specific protein 7b) regulates neuronal cell morphology by enhancing microtubule and actin filament assembly.

    PubMed

    Gotoh, Aina; Hidaka, Masafumi; Hirose, Keiko; Uchida, Takafumi

    2013-11-29

    Neurons undergo several morphological changes as a part of normal neuron maturation process. Alzheimer disease is associated with increased neuroproliferation and impaired neuronal maturation. In this study, we demonstrated that Gas7b (growth arrest specific protein 7b) expression in a neuronal cell line, Neuro 2A, induces cell maturation by facilitating formation of dendrite-like processes and/or filopodia projections and that Gas7b co-localizes with neurite microtubules. Molecular analysis was performed to evaluate whether Gas7b associates with actin filaments and microtubules, and the data revealed two novel roles of Gas7b in neurite outgrowth: we showed that Gas7b enhances bundling of several microtubule filaments and connects microtubules with actin filaments. These results suggest that Gas7b governs neural cell morphogenesis by enhancing the coordination between actin filaments and microtubules. We conclude that lower neuronal Gas7b levels may impact Alzheimer disease progression.

  15. Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins

    PubMed Central

    1994-01-01

    Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells. PMID:8051203

  16. Loss of Sarcomere-associated Formins Disrupts Z-line Organization, but does not Prevent Thin Filament Assembly in Caenorhabditis elegans Muscle

    PubMed Central

    Mi-Mi, Lei; Pruyne, David

    2015-01-01

    Members of the formin family of actin filament nucleation factors have been implicated in sarcomere formation, but precisely how these proteins affect sarcomere structure remains poorly understood. Of six formins in the simple nematode Caenorhabditis elegans, only FHOD-1 and CYK-1 contribute to sarcomere assembly in the worm's obliquely striated body-wall muscles. We analyze here the ultrastructure of body-wall muscle sarcomeres in worms with putative null fhod-1 and cyk-1 gene mutations. Contrary to a simple model that formins nucleate actin for thin filament assembly, formin mutant sarcomeres contain thin filaments. Rather, formin mutant sarcomeres are narrower and have deformed thin filament-anchoring Z-line structures. Thus, formins affect multiple aspects of sarcomere structure. PMID:26161293

  17. Cargo Transport by Two Coupled Myosin Va Motors on Actin Filaments and Bundles.

    PubMed

    Ali, M Yusuf; Vilfan, Andrej; Trybus, Kathleen M; Warshaw, David M

    2016-11-15

    Myosin Va (myoVa) is a processive, actin-based molecular motor essential for intracellular cargo transport. When a cargo is transported by an ensemble of myoVa motors, each motor faces significant physical barriers and directional challenges created by the complex actin cytoskeleton, a network of actin filaments and actin bundles. The principles that govern the interaction of multiple motors attached to the same cargo are still poorly understood. To understand the mechanical interactions between multiple motors, we developed a simple in vitro model in which two individual myoVa motors labeled with different-colored Qdots are linked via a third Qdot that acts as a cargo. The velocity of this two-motor complex was reduced by 27% as compared to a single motor, whereas run length was increased by only 37%, much less than expected from multimotor transport models. Therefore, at low ATP, which allowed us to identify individual motor steps, we investigated the intermotor dynamics within the two-motor complex. The randomness of stepping leads to a buildup of tension in the linkage between motors-which in turn slows down the leading motor-and increases the frequency of backward steps and the detachment rate. We establish a direct relationship between the velocity reduction and the distribution of intermotor distances. The analysis of run lengths and dwell times for the two-motor complex, which has only one motor engaged with the actin track, reveals that half of the runs are terminated by almost simultaneous detachment of both motors. This finding challenges the assumptions of conventional multimotor models based on consecutive motor detachment. Similar, but even more drastic, results were observed with two-motor complexes on actin bundles, which showed a run length that was even shorter than that of a single motor.

  18. Brownian Ratchets in Biophysics: from Diffusing Phospholipids to Polymerizing Actin Filaments

    NASA Astrophysics Data System (ADS)

    van Oudenaarden, Alexander

    2000-03-01

    In the 'Feynman Lectures on Physics' Feynman introduces a mechanical ratchet and pawl subjected to thermal fluctuations to demonstrate the impossibility to violate the second law of thermodynamics. Since this introduction the Brownian ratchet has evolved from Gedanken experiments to real experiments in the interdisciplinary sciences such as biophysics and biochemistry. In this symposium I will present two experiments in which the concept Brownian ratchet is of key importance. The first experiment addresses a so-called geometrical Brownian ratchet [1]. This ratchet consists of a two-dimensional microfabricated periodic array of asymmetric diffusion barriers. As an experimental realization of a two-dimensional fluid of Brownian particles, a bilayer of phospholipid molecules is used. I will demonstrate that the geometrical Brownian ratchet can be used as a molecular sieve to separate mixtures of membrane molecules without the need to extract them from the membrane. In the second experiment I explore the spontaneous symmetry breaking of polymerizing actin networks [2]. Small submicron size beads coated uniformly with a protein that catalyzes actin polymerization, are initially surrounded by a symmetrical cloud of actin filaments. This symmetry can be broken spontaneously after which the beads undergo directional motion with constant velocity. I will present a simple stochastic theory, in which each filament is modeled as an elastic Brownian ratchet that qualitatively reproduces the experimental results. The presence of the bead couples the dynamics of different filaments which results in a complex collective system of interacting Brownian ratchets that exhibits an emergent symmetry breaking behavior. [1] A. van Oudenaarden and S. G. Boxer, Science 285, 1046 (1999). [2] A. van Oudenaarden and J. A. Theriot, Nature Cell Biology 1, 493 (1999).

  19. Effects of asymmetric nanostructures on the extinction difference properties of actin biomolecules and filaments

    PubMed Central

    Khoo, E. H.; Leong, Eunice S. P.; Wu, S. J.; Phua, W. K.; Hor, Y. L.; Liu, Y. J.

    2016-01-01

    In this paper, symmetric and asymmetric tapering on the arms of the gammadion nanostructure is proposed to enhance both local field distribution and extinction difference (ED). The asymmetric tapered gammadion with tapering fraction (TF) of 0.67 is seen to have the largest ED and spatial local field distribution, producing a large wavelength shift of more than 50 percent as compared to the untapered gammadion nanostructures when immersed in a solution of actin molecules and filaments. The optical chirality, ζ shows that the larger local field amplitudes produced by the asymmetric designs increases the rate of chiral molecules excitation. This enhanced field is strongly rotating and highly sensitive to single molecules and larger filaments. Here, we show that the ED, optical chirality, sensitivity and rate of chiral molecules excitation can be improved by incorporating asymmetric designs into chiral gammadion nanostructures through tapering. PMID:26792371

  20. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M.

    2014-03-10

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.

  1. DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes

    PubMed Central

    Sun, Ming-Hong; Yang, Mo; Xie, Feng-Yun; Wang, Wei; Zhang, Lili; Shen, Wei; Yin, Shen

    2017-01-01

    As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells. PMID:28099474

  2. Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments.

    PubMed

    Okubo, Hiroyuki; Iwai, Masanori; Iwai, Sosuke; Chaen, Shigeru

    2010-05-28

    Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations.

  3. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin.

    PubMed

    Chaudhry, Faisal; Breitsprecher, Dennis; Little, Kristin; Sharov, Grigory; Sokolova, Olga; Goode, Bruce L

    2013-01-01

    Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

  4. Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis

    PubMed Central

    1994-01-01

    The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers. PMID:8034744

  5. Structure, sarcomeric organization, and thin filament binding of cardiac myosin-binding protein-C.

    PubMed

    Craig, Roger; Lee, Kyoung Hwan; Mun, Ji Young; Torre, Iratxe; Luther, Pradeep K

    2014-03-01

    Myosin-binding protein-C (MyBP-C) is an accessory protein of the myosin filaments of vertebrate striated muscle. In the heart, it plays a key role in modulating contractility in response to β-adrenergic stimulation. Mutations in the cardiac isoform (cMyBP-C) are a leading cause of inherited hypertrophic cardiomyopathy. Understanding cMyBP-C function and its role in disease requires knowledge of the structure of the molecule, its organization in the sarcomere, and its interactions with other sarcomeric proteins. Here we review the main structural features of this modular, elongated molecule and the properties of some of its key domains. We describe observations suggesting that the bulk of the molecule extends perpendicular to the thick filament, enabling it to reach neighboring thin filaments in the sarcomere. We review structural and functional evidence for interaction of its N-terminal domains with actin and how this may modulate thin filament activation. We also discuss the effects that phosphorylation of cMyBP-C has on some of these structural features and how this might relate to cMyBP-C function in the beating heart.

  6. Differential effects of thin and thick filament disruption on zebrafish smooth muscle regulatory proteins

    PubMed Central

    Davuluri, G.; Seiler, C.; Abrams, J.; Soriano, A. J.; Pack, M.

    2013-01-01

    Background The smooth muscle actin binding proteins Caldesmon and Tropomyosin (Tm) promote thin filament assembly by stabilizing actin polymerization, however, whether filament assembly affects either the stability or activation of these and other smooth muscle regulatory proteins is not known. Methods Measurement of smooth muscle regulatory protein levels in wild type zebrafish larvae following antisense knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins, and in colourless mutants that lack enteric nerves. Comparison of intestinal peristalsis in wild type and colourless larvae. Key Results Knockdown of Acta2 led to reduced levels of phospho-Caldesmon and Tm. Total Caldesmon and phospho-myosin light chain (p-Mlc) levels were unaffected. Knockdown of Myh11 had no effect on the levels of either of these proteins. Phospho-Caldesmon and p-Mlc levels were markedly reduced in colourless mutants that have intestinal motility comparable with wild type larvae. Conclusions & Inferences These in vivo findings provide new information regarding the activation and stability of smooth muscle regulatory proteins in zebrafish larvae and their role in intestinal peristalsis in this model organism. PMID:20591105

  7. Characterization and regulation of an additional actin-filament-binding site in large isoforms of the stereocilia actin-bundling protein espin.

    PubMed

    Zheng, Lili; Beeler, Dina M; Bartles, James R

    2014-03-15

    The espin actin-bundling proteins, which are produced as isoforms of different sizes from a single gene, are required for the growth of hair cell stereocilia. We have characterized an additional actin-filament-binding site present in the extended amino-termini of large espin isoforms. Constitutively active in espin 2, the site increased the size of actin bundles formed in vitro and inhibited actin fluorescence recovery in microvilli. In espin 1, which has an N-terminal ankyrin repeat domain, the site was autoinhibited by binding between the ankyrin repeat domain and a peptide near the actin-binding site. Deletion of this peptide from espin 1 activated its actin-binding site. The peptide resembled tail homology domain I of myosin III, a ligand of the ankyrin repeat domain localized with espin 1 at the tip of stereocilia. A myosin III tail homology domain I peptide, but not scrambled control peptides, inhibited internal binding of the ankyrin repeat domain and released the espin 1 actin-binding site from autoinhibition. Thus, this regulation could result in local activation of the additional actin-binding site of espin 1 by myosin III in stereocilia.

  8. Migration of Nucleocapsids in Vesicular Stomatitis Virus-Infected Cells Is Dependent on both Microtubules and Actin Filaments

    PubMed Central

    Yacovone, Shalane K.; Smelser, Amanda M.; Macosko, Jed C.; Holzwarth, George; Ornelles, David A.

    2016-01-01

    ABSTRACT The distribution of vesicular stomatitis virus (VSV) nucleocapsids in the cytoplasm of infected cells was analyzed by scanning confocal fluorescence microscopy using a newly developed quantitative approach called the border-to-border distribution method. Nucleocapsids were located near the cell nucleus at early times postinfection (2 h) but were redistributed during infection toward the edges of the cell. This redistribution was inhibited by treatment with nocodazole, colcemid, or cytochalasin D, indicating it is dependent on both microtubules and actin filaments. The role of actin filaments in nucleocapsid mobility was also confirmed by live-cell imaging of fluorescent nucleocapsids of a virus containing P protein fused to enhanced green fluorescent protein. However, in contrast to the overall redistribution in the cytoplasm, the incorporation of nucleocapsids into virions as determined in pulse-chase experiments was dependent on the activity of actin filaments with little if any effect on inhibition of microtubule function. These results indicate that the mechanisms by which nucleocapsids are transported to the farthest reaches of the cell differ from those required for incorporation into virions. This is likely due to the ability of nucleocapsids to follow shorter paths to the plasma membrane mediated by actin filaments. IMPORTANCE Nucleocapsids of nonsegmented negative-strand viruses like VSV are assembled in the cytoplasm during genome RNA replication and must migrate to the plasma membrane for assembly into virions. Nucleocapsids are too large to diffuse in the cytoplasm in the time required for virus assembly and must be transported by cytoskeletal elements. Previous results suggested that microtubules were responsible for migration of VSV nucleocapsids to the plasma membrane for virus assembly. Data presented here show that both microtubules and actin filaments are responsible for mobility of nucleocapsids in the cytoplasm, but that actin filaments

  9. Correlation of expression of the actin filament-bundling protein espin with stereociliary bundle formation in the developing inner ear.

    PubMed

    Li, Huawei; Liu, Hong; Balt, Steve; Mann, Sabine; Corrales, C Eduardo; Heller, Stefan

    2004-01-01

    The vertebrate hair cell is named for its stereociliary bundle or hair bundle that protrudes from the cell's apical surface. Hair bundles mediate mechanosensitivity, and their highly organized structure plays a critical role in mechanoelectrical transduction and amplification. The prototypical hair bundle is composed of individual stereocilia, 50-300 in number, depending on the animal species and on the type of hair cell. The assembly of stereocilia, in particular, the formation during development of individual rows of stereocilia with descending length, has been analyzed in great morphological detail. Electron microscopic studies have demonstrated that stereocilia are filled with actin filaments that are rigidly cross-linked. The growth of individual rows of stereocilia is associated with the addition of actin filaments and with progressively increasing numbers of cross-bridges between actin filaments. Recently, a mutation in the actin filament-bundling protein espin has been shown to underlie hair bundle degeneration in the deaf jerker mouse, subsequently leading to deafness. Our study was undertaken to investigate the appearance and developmental expression of espin in chicken inner ear sensory epithelia. We found that the onset of espin expression correlates with the initiation and growth of stereocilia bundles in vestibular and cochlear hair cells. Intense espin immunolabeling of stereocilia was colocalized with actin filament staining in all types of hair cells at all developmental stages and in adult animals. Our analysis of espin as a molecular marker for actin filament cross-links in stereocilia is in full accordance with previous morphological studies and implicates espin as an important structural component of hair bundles from initiation of bundle assembly to mature chicken hair cells.

  10. Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules

    PubMed Central

    Han, Xiaofan; Li, Pin; Yang, Zhenghao; Huang, Xiaoshuai; Wei, Guoqin; Sun, Yujie; Kang, Xuya; Hu, Xueting; Deng, Qiuping; Chen, Liangyi; He, Aibin; Huo, Yingqing; Li, Dong; Betzig, Eric; Luo, Jincai

    2017-01-01

    Endothelial exocytosis of Weibel–Palade body (WPB) is one of the first lines of defence against vascular injury. However, the mechanisms that control WPB exocytosis in the final stages (including the docking, priming and fusion of granules) are poorly understood. Here we show that the focal adhesion protein zyxin is crucial in this process. Zyxin downregulation inhibits the secretion of von Willebrand factor (VWF), the most abundant cargo in WPBs, from human primary endothelial cells (ECs) induced by cAMP agonists. Zyxin-deficient mice exhibit impaired epinephrine-stimulated VWF release, prolonged bleeding time and thrombosis, largely due to defective endothelial secretion of VWF. Using live-cell super-resolution microscopy, we visualize previously unappreciated reorganization of pre-existing actin filaments around WPBs before fusion, dependent on zyxin and an interaction with the actin crosslinker α-actinin. Our findings identify zyxin as a physiological regulator of endothelial exocytosis through reorganizing local actin network in the final stage of exocytosis. PMID:28256511

  11. Sarcomeric thick and thin filament overlap influences postmortem proteolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The interaction between sarcomere length (SL) and proteolysis on meat tenderness is not clear. Indeed, the extent of thick and thin filament overlap alters actomyosin binding and may alter substrate availability during aging. The objective of this study was to determine the influence of sarcomere le...

  12. The rearrangement of filamentous actin in mossy fiber synapses in pentylenetetrazol-kindled C57BL/6 mice.

    PubMed

    Zhang, Yan-Feng; Li, Shu-Lei; Xiong, Tian-Qing; Yang, Li-Bin; Li, Yong-Nan; Tan, Bai-Hong; Liu, Qun; Li, Yan-Chao

    2014-01-01

    Chemical kindling, as an experimental model of epileptogenesis, is induced by repetitive administration of subconvulsive amount of excitatory drugs. Kindled mice do not typically display spontaneous recurrent seizures, but are instead characterized by enhanced seizure susceptibility to convulsive stimulations. In order to provide insights into the aberrant synaptic plasticity during kindling, this study investigated the effect of pentylenetetrazol (PTZ) kindling on filamentous actin (F-actin) in mossy fiber synapses in C57BL/6 mice. Phalloidin labeling of F-actin showed that F-actin puncta were increased in number in the stratum lucidum of CA3 region in the hippocampus after kindling. The rearrangement of F-actin seemed to occur presynaptically, since synapsin I, a specific marker for mossy fiber terminals, was also up-regulated. Such subtle structural modifications occurring in the synapses are thought to contribute to the long-lasting increased sensitivity in the PTZ-kindled C57BL/6 mice.

  13. Reaction-diffusion waves of actin filament polymerization/depolymerization in Dictyostelium pseudopodium extension and cell locomotion.

    PubMed

    Vicker, M G

    2000-04-14

    Cell surface movements and the intracellular spatial patterns and dynamics of actin filament (F-actin) were investigated in living and formalin-fixed cells of Dictyostelium discoideum by confocal microscopy. Excitation waves of F-actin assembly developed and propagated several micrometers at up to 26 microm/min in cells which had been intracellularly loaded with fluorescently labeled actin monomer. Wave propagation and extinction corresponded with the initiation and attenuation of pseudopodium extension and cell advance, respectively. The identification of chemical waves was supported by the ring, sphere, spiral and scroll wave patterns, which were observed in the extensions of fixed cells stained with phalloidin-rhodamine, and by the similar, asymmetrical [F-actin] distribution in wavefronts in living and fixed cells. These F-actin patterns and dynamics in Dictyostelium provide evidence for a new supramolecular state of actin, which propagates as a self-organized, reaction-diffusion wave of reversible F-actin assembly and affects pseudopodium extension. Actin's properties of oscillation and self-organization might also fundamentally determine the nature of the eukaryotic cell's reactions of adaptation, timing and signal response.

  14. Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly.

    PubMed

    Freeman, N L; Field, J

    2000-02-01

    Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.

  15. Kinetic heterogeneity of F-actin polymers. Further evidence that the elongation reaction may occur through condensation of the actin filaments with small aggregates.

    PubMed Central

    Grazi, E; Magri, E

    1987-01-01

    We have shown that F-actin, polymerized in 50 mM-KCl at 20 degrees C and pH 8.0, can be resolved by centrifugation into two polymer populations, which differ morphologically as well as kinetically. The first population represents about 10% of the overall polymer and is composed of small amorphous aggregates. It rapidly exchanges the bound nucleotide with free ATP in the medium, either directly or through the monomers. The second population is composed of long actin filaments. These are labelled by free ATP in the medium only through condensation with labelled small amorphous aggregates. Images Fig. 1. PMID:3435480

  16. Direct observation of the uncapping of capping protein-capped actin filaments by CARMIL homology domain 3.

    PubMed

    Fujiwara, Ikuko; Remmert, Kirsten; Hammer, John A

    2010-01-22

    Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of approximately 90% at approximately 250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from approximately 30 min without mCAH3 to approximately 10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments.

  17. Direct Observation of the Uncapping of Capping Protein-capped Actin Filaments by CARMIL Homology Domain 3*

    PubMed Central

    Fujiwara, Ikuko; Remmert, Kirsten; Hammer, John A.

    2010-01-01

    Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of ∼90% at ∼250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from ∼30 min without mCAH3 to ∼10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments. PMID:19926785

  18. Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface

    PubMed Central

    1995-01-01

    The thermodynamic basis for actin-based motility of Listeria monocytogenes has been investigated using cytoplasmic extracts of Xenopus eggs, initially developed by Theriot et al. (Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Cell. 76:505-517) as an in vitro cell-free system. A large proportion (75%) of actin was found unpolymerized in the extracts. The amount of unassembled actin (12 microM) is accounted for by the sequestering functions of T beta 4Xen (20 microM) and profilin (5 microM), the barbed ends being capped. Movement of Listeria was not abolished by depletion of over 99% of the endogenous profilin. The proline-rich sequences of ActA are unlikely to be the target of profilin. All data support the view that actin assembly at the rear of Listeria results from a local shift in steady state due to a factor, keeping filaments uncapped, bound to the surface of the bacterium, while barbed ends are capped in the bulk cytoplasm. Movement is controlled by the energetic difference (i.e., the difference in critical concentration) between the two ends of the filaments, hence a constant ATP supply and the presence of barbed end capped F-actin in the medium are required to buffer free G-actin at a high concentration. The role of membrane components is demonstrated by the facts that: (a) Listeria movement can be reconstituted in the resuspended pellets of high speed-centrifuged extracts that are enriched in membranes; (b) Actin-based motility of endogenous vesicles, exhibiting the same rocketing movement as Listeria, can be observed in the extracts. PMID:7615635

  19. Specific Transformation of Assembly with Actin Filaments and Molecular Motors in a Cell-Sized Self-Emerged Liposome

    NASA Astrophysics Data System (ADS)

    Takiguchi, Kingo; Negishi, Makiko; Tanaka-Takiguchi, Yohko; Hayashi, Masahito; Yoshikawa, Kenichi

    2014-12-01

    Eukaryotes, by the same combination of cytoskeleton and molecular motor, for example actin filament and myosin, can generate a variety of movements. For this diversity, the organization of biological machineries caused by the confinement and/or crowding effects of internal living cells, may play very important roles.

  20. Vimentin intermediate filaments control actin stress fiber assembly through GEF-H1 and RhoA

    PubMed Central

    Peränen, Johan; Schaible, Niccole; Cheng, Fang; Eriksson, John E.; Krishnan, Ramaswamy

    2017-01-01

    ABSTRACT The actin and intermediate filament cytoskeletons contribute to numerous cellular processes, including morphogenesis, cytokinesis and migration. These two cytoskeletal systems associate with each other, but the underlying mechanisms of this interaction are incompletely understood. Here, we show that inactivation of vimentin leads to increased actin stress fiber assembly and contractility, and consequent elevation of myosin light chain phosphorylation and stabilization of tropomyosin-4.2 (see Geeves et al., 2015). The vimentin-knockout phenotypes can be rescued by re-expression of wild-type vimentin, but not by the non-filamentous ‘unit length form’ vimentin, demonstrating that intact vimentin intermediate filaments are required to facilitate the effects on the actin cytoskeleton. Finally, we provide evidence that the effects of vimentin on stress fibers are mediated by activation of RhoA through its guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2). Vimentin depletion induces phosphorylation of the microtubule-associated GEF-H1 on Ser886, and thereby promotes RhoA activity and actin stress fiber assembly. Taken together, these data reveal a new mechanism by which intermediate filaments regulate contractile actomyosin bundles, and may explain why elevated vimentin expression levels correlate with increased migration and invasion of cancer cells. PMID:28096473

  1. The ALP-Enigma protein ALP-1 functions in actin filament organization to promote muscle structural integrity in Caenorhabditis elegans.

    PubMed

    Han, Hsiao-Fen; Beckerle, Mary C

    2009-05-01

    Mutations that affect the Z-disk-associated ALP-Enigma proteins have been linked to human muscular and cardiac diseases. Despite their clear physiological significance for human health, the mechanism of action of ALP-Enigma proteins is largely unknown. In Caenorhabditis elegans, the ALP-Enigma protein family is encoded by a single gene, alp-1; thus C. elegans provides an excellent model to study ALP-Enigma function. Here we present a molecular and genetic analysis of ALP-Enigma function in C. elegans. We show that ALP-1 and alpha-actinin colocalize at dense bodies where actin filaments are anchored and that the proper localization of ALP-1 at dense bodies is dependent on alpha-actinin. Our analysis of alp-1 mutants demonstrates that ALP-1 functions to maintain actin filament organization and participates in muscle stabilization during contraction. Reducing alpha-actinin activity enhances the actin filament phenotype of the alp-1 mutants, suggesting that ALP-1 and alpha-actinin function in the same cellular process. Like alpha-actinin, alp-1 also interacts genetically with a connectin/titin family member, ketn-1, to provide mechanical stability for supporting body wall muscle contraction. Taken together, our data demonstrate that ALP-1 and alpha-actinin function together to stabilize actin filaments and promote muscle structural integrity.

  2. Demonstration of actin filament stress fibers in microvascular endothelial cells in situ.

    PubMed

    Nehls, V; Drenckhahn, D

    1991-07-01

    We have developed a method for immunostaining the microvascular tree of rat mesenteric windows in situ. The procedure consists of three steps, i.e., mild fixation with formaldehyde, controlled proteolytic digestion of the mesothelial layer, and permeabilization with acetone. Discrimination between different microvascular segments was possible by double-fluorescent staining with antibodies to the smooth muscle isoform of alpha-actin and to nonmuscle myosin from platelets. Antibodies to nonmuscle myosin labeled numerous longitudinally oriented cables in endothelial cells of all microvascular segments (arterioles, metarterioles, pre-, mid-, and postcapillaries, small venules). Occasionally, the myosin-containing cables displayed the interrupted sarcomere-like staining pattern that is diagnostic for stress fibers. In contrast, staining of actin filaments with phalloidin-rhodamin resulted in a noninterrupted, continuous fluorescence of the stress fibers. A possible functional role of microvascular endothelial stress fibers is to serve as a tensile cytoskeletal scaffold that stabilizes the tubular, three-dimensional geometry of microvessels and, in addition, to help the endothelium resist the shear forces created by blood flow and by collision with red and white blood cells.

  3. Oxygen radicals production and actin filament disruption in bivalve haemocytes treated with benzo(a)pyrene.

    PubMed

    Gómez-Mendikute, Amagoia; Etxeberria, Ainhoa; Olabarrieta, Igor; Cajaraville, Miren P

    2002-01-01

    Haemocytes play an essential role in the internal defence of molluscs. It has been reported that organic xenobiotics commonly found as pollutants in the marine environment impair defence capabilities of haemocytes. The purpose of the present study was to investigate the effects of benzo(a)pyrene [B(a)P] on the integrity of the actin cytoskeleton and on endocytosis in haemocytes and to see if these effects are related to generation of reactive oxygen species. Haemocytes were exposed in vitro to B(a)P (0.5-40 microg/ml) for 1 h. Cell viability (using 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide or XTT assay) indicated that selected doses were sublethal. Uptake of neutral red was significantly decreased in a dose-dependent manner in B(a)P-treated haemocytes. Distribution of actin filaments, labeled with rhodamine-conjugated phalloidin, was altered in haemocytes treated with 20 or 40 microg/ml B(a)P. These effects could be related to an increased production of superoxide anion during B(a)P metabolism, as detected by the nitroblue tetrazolium (NBT) reduction assay in haemocytes treated with > or = 10 microg/ml B(a)P.

  4. Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

    PubMed

    Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

    2015-04-24

    EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein.

  5. Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers.

    PubMed

    Losinno, Antonella D; Morales, Alfonsina; Fernández, Dario; Lopez, Luis A

    2012-05-01

    In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.

  6. Single-Molecule Discrimination within Dendritic Spines of Discrete Perisynaptic Sites of Actin Filament Assembly Driving Postsynaptic Reorganization

    NASA Astrophysics Data System (ADS)

    Blanpied, Thomas A.

    2013-03-01

    In the brain, the strength of synaptic transmission between neurons is principally set by the organization of proteins within the receptive, postsynaptic cell. Synaptic strength at an individual site of contact can remain remarkably stable for months or years. However, it also can undergo diverse forms of plasticity which change the strength at that contact independent of changes to neighboring synapses. Such activity-triggered neural plasticity underlies memory storage and cognitive development, and is disrupted in pathological physiology such as addiction and schizophrenia. Much of the short-term regulation of synaptic plasticity occurs within the postsynaptic cell, in small subcompartments surrounding the synaptic contact. Biochemical subcompartmentalization necessary for synapse-specific plasticity is achieved in part by segregation of synapses to micron-sized protrusions from the cell called dendritic spines. Dendritic spines are heavily enriched in the actin cytoskeleton, and regulation of actin polymerization within dendritic spines controls both basal synaptic strength and many forms of synaptic plasticity. However, understanding the mechanism of this control has been difficult because the submicron dimensions of spines limit examination of actin dynamics in the spine interior by conventional confocal microscopy. To overcome this, we developed single-molecule tracking photoactivated localization microscopy (smtPALM) to measure the movement of individual actin molecules within living spines. This revealed inward actin flow from broad areas of the spine plasma membrane, as well as a dense central core of heterogeneous filament orientation. The velocity of single actin molecules along filaments was elevated in discrete regions within the spine, notably near the postsynaptic density but surprisingly not at the endocytic zone which is involved in some forms of plasticity. We conclude that actin polymerization is initiated at many well-separated foci within

  7. Observation of Cell Shortening and Dynamic Changes of Actin Filaments during Cell Detachment from Thermoresponsive-Gelatin-Coated Substrate

    NASA Astrophysics Data System (ADS)

    Nagayama, Kazuaki; Matsumoto, Takeo

    We observed cell shortening and dynamic changes of actin filaments during detachment from the substrate by using a thermoresponsive-gelatin. The thermoresponsive-gelatin, a mixture of the poly(N-isopropylacrylamide) grafted gelatin (PNIPAAm-grafted-gelatin) and PNIPAAm, was coated on the glass bottom culture dishes. Rat aortic smooth muscle cells (SMC) expressed GFP-actin were cultured on the thermoresponsive-gelatin-coated dishes filled with serum free Dulbecco’s Modified Eagle’s medium (DMEM) or Ca2 + -Mg2 + -free Hank’s balanced salt solution (HBSS(-)). They adhered normally at 37°C, and became shortened and detached from the dishes when the ambient temperature was dropped below 34°C due to melting of the gelatin substrate. The shortening of SMCs was larger in DMEM (51.0 ±3.0%, mean ± SEM, n=14) than in HBSS(-) (35.8 ±3.6%, n=14). Actin filaments remained straight during detachment in HBSS(-), while in DMEM, they locally concentrated and disappeared at cell periphery. The shortening of SMCs upon detachment from ordinary plastic culture dishes by trypsinization was more than for both media. No significant difference was observed between the two, indicating detrimental effects of the trypsin. The present method was useful to study the cell contraction and the actin filament behavior during cell detachment for this causes minimal damage to cells.

  8. Cardiac thin filament regulation and the Frank-Starling mechanism.

    PubMed

    Kobirumaki-Shimozawa, Fuyu; Inoue, Takahiro; Shintani, Seine A; Oyama, Kotaro; Terui, Takako; Minamisawa, Susumu; Ishiwata, Shin'ichi; Fukuda, Norio

    2014-07-01

    The heart has an intrinsic ability to increase systolic force in response to a rise in ventricular filling (the Frank-Starling law of the heart). It is widely accepted that the length dependence of myocardial activation underlies the Frank-Starling law of the heart. Recent advances in muscle physiology have enabled the identification of the factors involved in length-dependent activation, viz., titin (connectin)-based interfilament lattice spacing reduction and thin filament "on-off" regulation, with the former triggering length-dependent activation and the latter determining the number of myosin molecules recruited to thin filaments. Patients with a failing heart have demonstrated reduced exercise tolerance at least in part via depression of the Frank-Starling mechanism. Recent studies revealed that various mutations occur in the thin filament regulatory proteins, such as troponin, in the ventricular muscle of failing hearts, which consequently alter the Frank-Starling mechanism. In this article, we review the molecular mechanisms of length-dependent activation, and the influence of troponin mutations on the phenomenon.

  9. The Interaction of Arp2/3 Complex with Actin: Nucleation, High Affinity Pointed End Capping, and Formation of Branching Networks of Filaments

    NASA Astrophysics Data System (ADS)

    Dyche Mullins, R.; Heuser, John A.; Pollard, Thomas D.

    1998-05-01

    The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μ M. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

  10. Formin and capping protein together embrace the actin filament in a ménage à trois

    PubMed Central

    Shekhar, Shashank; Kerleau, Mikael; Kühn, Sonja; Pernier, Julien; Romet-Lemonne, Guillaume; Jégou, Antoine; Carlier, Marie-France

    2015-01-01

    Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed. PMID:26564775

  11. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

    PubMed

    Pal Sharma, C; Goldmann, Wolfgang H

    2004-01-01

    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  12. Disorder profile of nebulin encodes a vernierlike position sensor for the sliding thin and thick filaments of the skeletal muscle sarcomere

    NASA Astrophysics Data System (ADS)

    Wu, Ming-Chya; Forbes, Jeffrey G.; Wang, Kuan

    2016-06-01

    Nebulin is an about 1 μ m long intrinsically disordered scaffold for the thin filaments of skeletal muscle sarcomere. It is a multifunctional elastic protein that wraps around actin filament, stabilizes thin filaments, and regulates Ca-dependent actomyosin interactions. This study investigates whether the disorder profile of nebulin might encode guidelines for thin and thick filament interactions in the sarcomere of the skeletal muscle. The question was addressed computationally by analyzing the predicted disorder profile of human nebulin (6669 residues, ˜200 actin-binding repeats) by pondr and the periodicity of the A-band stripes (reflecting the locations of myosin-associated proteins) in the electron micrographs of the sarcomere. Using the detrended fluctuation analysis, a scale factor for the A-band stripe image data with respect to the nebulin disorder profile was determined to make the thin and thick filaments aligned to have maximum correlation. The empirical mode decomposition method was then applied to identify hidden periodicities in both the nebulin disorder profile and the rescaled A-band data. The decomposition reveals three characteristic length scales (45 nm, 100 nm, and 200 nm) that are relevant for correlational analysis. The dynamical cross-correlation analyses with moving windows at various sarcomere lengths depict a vernierlike design for both periodicities, thus enabling nebulin to sense position and fine tune sarcomere overlap. This shows that the disorder profile of scaffolding proteins may encode a guideline for cellular architecture.

  13. Myosin IIIa boosts elongation of stereocilia by transporting espin 1 to the plus ends of actin filaments.

    PubMed

    Salles, Felipe T; Merritt, Raymond C; Manor, Uri; Dougherty, Gerard W; Sousa, Aurea D; Moore, Judy E; Yengo, Christopher M; Dosé, Andréa C; Kachar, Bechara

    2009-04-01

    Two proteins implicated in inherited deafness, myosin IIIa, a plus-end-directed motor, and espin, an actin-bundling protein containing the actin-monomer-binding motif WH2, have been shown to influence the length of mechanosensory stereocilia. Here we report that espin 1, an ankyrin repeat-containing isoform of espin, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that combined overexpression of these proteins causes greater elongation of stereocilia, compared with overexpression of either myosin IIIa alone or espin 1 alone. When these two proteins were co-expressed in the fibroblast-like COS-7 cell line they induced a tenfold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role of myosin IIIa and espin 1 in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.

  14. Emerging roles of sumoylation in the regulation of actin, microtubules, intermediate filaments, and septins

    PubMed Central

    Alonso, Annabel; Greenlee, Matt; Matts, Jessica; Kline, Jake; Davis, Kayla J.

    2015-01-01

    Sumoylation is a powerful regulatory system that controls many of the critical processes in the cell, including DNA repair, transcriptional regulation, nuclear transport, and DNA replication. Recently, new functions for SUMO have begun to emerge. SUMO is covalently attached to components of each of the four major cytoskeletal networks, including microtubule‐associated proteins, septins, and intermediate filaments, in addition to nuclear actin and actin‐regulatory proteins. However, knowledge of the mechanisms by which this signal transduction system controls the cytoskeleton is still in its infancy. One story that is beginning to unfold is that SUMO may regulate the microtubule motor protein dynein by modification of its adaptor Lis1. In other instances, cytoskeletal elements can both bind to SUMO non‐covalently and also be conjugated by it. The molecular mechanisms for many of these new functions are not yet clear, but are under active investigation. One emerging model links the function of MAP sumoylation to protein degradation through SUMO‐targeted ubiquitin ligases, also known as STUbL enzymes. Other possible functions for cytoskeletal sumoylation are also discussed. © 2015 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:26033929

  15. Crystallization of fluorescent quantum dots within a three-dimensional bio-organic template of actin filaments and lipid membranes.

    PubMed

    Henry, Etienne; Dif, Aurélien; Schmutz, Marc; Legoff, Loic; Amblard, François; Marchi-Artzner, Valérie; Artzner, Franck

    2011-12-14

    Biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures. We demonstrate the ability of a self-assembled three-dimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots. Functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5×18.9×35.5 nm3, are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a large range of distance parameters.

  16. Thin-Filament Pyrometry Developed for Measuring Temperatures in Flames

    NASA Technical Reports Server (NTRS)

    Sunderland, Peter B.

    2004-01-01

    Many valuable advances in combustion science have come from observations of microgravity flames. This research is contributing to the improved efficiency and reduced emissions of practical combustors and is benefiting terrestrial and spacecraft fire safety. Unfortunately, difficulties associated with microgravity have prevented many types of measurements in microgravity flames. In particular, temperature measurements in flames are extremely important but have been limited in microgravity. A novel method of measuring temperatures in microgravity flames is being developed in-house at the National Center for Microgravity Research and the NASA Glenn Research Center and is described here. Called thin-filament pyrometry, it involves using a camera to determine the local gas temperature from the intensity of inserted fibers glowing in a flame. It is demonstrated here to provide accurate measurements of gas temperatures in a flame simultaneously at many locations. The experiment is shown. The flame is a laminar gas jet diffusion flame fueled by methane (CH4) flowing from a 14-mm round burner at a pressure of 1 atm. A coflowing stream of air is used to prevent flame flicker. Nine glowing fibers are visible. These fibers are made of silicon carbide (SiC) and have a diameter of 15 m (for comparison, the average human hair is 75 m in diameter). Because the fibers are so thin, they do little to disturb the flame and their temperature remains close to that of the local gas. The flame and glowing filaments were imaged with a digital black-and-white video camera. This camera has an imaging area of 1000 by 1000 pixels and a wide dynamic range of 12 bits. The resolution of the camera and optics was 0.1 mm. Optical filters were placed in front of the camera to limit incoming light to 750, 850, 950, and 1050 nm. Temperatures were measured in the same flame in the absence of fibers using 50-m Btype thermocouples. These thermocouples provide very accurate temperatures, but they

  17. Cooperative regulation of myosin-S1 binding to actin filaments by a continuous flexible Tm-Tn chain.

    PubMed

    Mijailovich, Srboljub M; Kayser-Herold, Oliver; Li, Xiaochuan; Griffiths, Hugh; Geeves, Michael A

    2012-12-01

    The regulation of striated muscle contraction involves cooperative interactions between actin filaments, myosin-S1 (S1), tropomyosin (Tm), troponin (Tn), and calcium. These interactions are modeled by treating overlapping tropomyosins as a continuous flexible chain (CFC), weakly confined by electrostatic interactions with actin. The CFC is displaced locally in opposite directions on the actin surface by the binding of either S1 or Troponin I (TnI) to actin. The apparent rate constants for myosin and TnI binding to and detachment from actin are then intrinsically coupled via the CFC model to the presence of neighboring bound S1s and TnIs. Monte Carlo simulations at prescribed values of the CFC stiffness, the CFC's degree of azimuthal confinement, and the angular displacements caused by the bound proteins were able to predict the stopped-flow transients of S1 binding to regulated F-actin. The transients collected over a large range of calcium concentrations could be well described by adjusting a single calcium-dependent parameter, the rate constant of TnI detachment from actin, k(-I). The resulting equilibrium constant K(B) ≡ 1/K(I) varied sigmoidally with the free calcium, increasing from 0.12 at low calcium (pCa >7) to 12 at high calcium (pCa <5.5) with a Hill coefficient of ~2.15. The similarity of the curves for excess-actin and excess-myosin data confirms their allosteric relationship. The spatially explicit calculations confirmed variable sizes for the cooperative units and clustering of bound myosins at low calcium concentrations. Moreover, inclusion of negative cooperativity between myosin units predicted the observed slowing of myosin binding at excess-myosin concentrations.

  18. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

    PubMed

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W; Gelfand, Vladimir I

    2014-07-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

  19. A tridimensional view of the organization of actin filaments in the central nervous system by use of fluorescent photooxidation.

    PubMed

    Capani, Francisco; Saraceno, Ezequiel; Boti, Valeria Romina; Aon-Bertolino, Laura; Fernández, Juan Carlos; Gato, Fernándo; Kruse, Maria Sol; Krause, Maria Sol; Giraldez, Lisandro; Ellisman, Mark H; Coirini, Héctor

    2008-04-01

    Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.

  20. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells

    PubMed Central

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830–840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250–1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and

  1. Measuring the flexural rigidity of actin filaments and microtubules from their thermal fluctuating shapes: A new perspective

    NASA Astrophysics Data System (ADS)

    Jia, Kangyu; Liu, Xiaohu

    Actin filaments and microtubules are important components of cytoskeletal networks and show both active and passive dynamic mechanical behaviors. Measuring the mechanical properties of individual filament can not only help us understand the mechanisms behind the complex dynamic behaviors, but also provide parameters that are needed to calibrate biological piconewton forcemeters. Although many methods have been proposed, the values of flexural rigidity reported in literature are still quite different for both actin filaments and microtubules. In this paper, a new formulation based on mode analysis of the thermal fluctuating shapes and principle of virtual work has been proposed, where both the linear and nonlinear assumptions are considered. What's more, following previous inspiring works, both the effects of sampling time interval and hydrodynamics are taken into account in our model. When applied to the experiment data in literature and the simulation data generated by finite element method software, our method gives good results and show an advantage over the previous methods. Besides, we suggest that the inconformity of the flexural rigidity in literature might be caused by the different sampling time intervals and hydrodynamic wall effects in experiments.

  2. Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.

    PubMed Central

    Omata, W; Shibata, H; Li, L; Takata, K; Kojima, I

    2000-01-01

    Actin-based cytoskeletons have been implicated in insulin-stimulated glucose transport and translocation of the insulin-regulated glucose transporter, GLUT4, from the intracellular pool to the plasma membrane. However, most previous studies were done using adherent cell systems such as L6 myotubes and 3T3-L1 adipocytes, and very little information is available on the significance of the actin filaments to the insulin action in isolated adipocytes, a widely used experimental system. In the present study, we investigated the physiological role of actin filaments in the subcellular trafficking of GLUT4 in isolated rat adipocytes. We first compared the effects of two actin-disrupting reagents, latrunculin A and cytochalasin D, on the organization of the actin filaments as well as on the insulin action on glucose transport by laser confocal microscopy combined with biochemical analysis of the insulin action. Treatment of the cells with latrunculin A induced dose- and time-dependent disappearance of the filamentous actin, which correlated very well with inhibition of the insulin effect on glucose transport. Although cytochalasin D at 50 microM significantly inhibited insulin-stimulated glucose transport, it was not effective in disassembly of the actin filaments; rather, many intense punctate signals were observed in cytochalasin D-treated cells. In the actin-disrupted adipocytes treated with latrunculin A, insulin-induced GLUT4 translocation was inhibited completely. In addition, latrunculin A remarkably inhibited both insulin-induced glucose transport and GLUT4 translocation in the presense of D(k)-(62-85), a potent inhibitor of GLUT4 endocytosis, suggesting that intactness of the actin filaments was necessary for insulin-induced exocytosis of the GLUT4-containing vesicles. On the other hand, latrunculin A showed little inhibitory effect on either endocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4 or decay of the glucose transport activity after addition of

  3. Drosophila Homologues of Adenomatous Polyposis Coli (APC) and the Formin Diaphanous Collaborate by a Conserved Mechanism to Stimulate Actin Filament Assembly*

    PubMed Central

    Jaiswal, Richa; Stepanik, Vince; Rankova, Aneliya; Molinar, Olivia; Goode, Bruce L.; McCartney, Brooke M.

    2013-01-01

    Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla. PMID:23558679

  4. Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

    PubMed Central

    Zhou, Zhenzhen; Shi, Haifan; Chen, Binqing; Zhang, Ruihui; Huang, Shanjin; Fu, Ying

    2015-01-01

    Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca2+ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth. PMID:25804540

  5. Biogenesis of actin-like bacterial cytoskeletal filaments destined for positioning prokaryotic magnetic organelles.

    PubMed

    Pradel, Nathalie; Santini, Claire-Lise; Bernadac, Alain; Fukumori, Yoshihiro; Wu, Long-Fei

    2006-11-14

    Magnetosomes comprise a magnetic nanocrystal surrounded by a lipid bilayer membrane. These unique prokaryotic organelles align inside magnetotactic bacterial cells and serve as an intracellular compass allowing the bacteria to navigate along the geomagnetic field in aquatic environments. Cryoelectron tomography of Magnetospirillum strains has revealed that the magnetosome chain is surrounded by a network of filaments that may be composed of MamK given that the filaments are absent in the mamK mutant cells. The process of the MamK filament assembly is unknown. Here we prove the authenticity of the MamK filaments and show that MamK exhibits linear distribution inside Magnetospirillum sp. cells even in the area without magnetosomes. The mamK gene alone is sufficient to direct the synthesis of straight filaments in Escherichia coli, and one extremity of the MamK filaments is located at the cellular pole. By using dual fluorescent labeling of MamK, we found that MamK nucleates at multiple sites and assembles into mosaic filaments. Time-lapse experiments reveal that the assembly of the MamK filaments is a highly dynamic and kinetically asymmetrical process. MamK bundles might initiate the formation of a new filament or associate to one preexistent filament. Our results demonstrate the mechanism of biogenesis of prokaryotic cytoskeletal filaments that are structurally and functionally distinct from the known MreB and ParM filaments. In addition to positioning magnetosomes, other hypothetical functions of the MamK filaments in magnetotaxis might include anchoring magnetosomes and being involved in magnetic reception.

  6. A coat of filamentous actin prevents clustering of late-endosomal vacuoles in vivo.

    PubMed

    Drengk, Anja; Fritsch, Jürgen; Schmauch, Christian; Rühling, Harald; Maniak, Markus

    2003-10-14

    The endocytic pathway depends on the actin cytoskeleton. Actin contributes to internalization at the plasma membrane and to subsequent trafficking steps like propulsion through the cytoplasm, fusion of phagosomes with early endosomes, and transport from early to late endosomes. In vitro studies with mammalian endosomes and yeast vacuoles implicate actin in membrane fusion. Here, we investigate the function of the actin coat that surrounds late endosomes in Dictyostelium. Latrunculin treatment leads to aggregation of these endosomes into grape-like clusters and completely blocks progression of endocytic marker. In addition, the cells round up and stop moving. Because this drug treatment perturbs all actin assemblies in the cell simultaneously, we used a novel targeting approach to specifically study the function of the cytoskeleton in one subcellular location. To this end, we constructed a hybrid protein targeting cofilin, an actin depolymerizing protein, to late endosomes. As a consequence, the endosomal compartments lost their actin coats and aggregated, but these cells remained morphologically normal, and the kinetics of endocytic marker trafficking were unaltered. Therefore, the actin coat prevents the clustering of endosomes, which could be one safeguard mechanism precluding their docking and fusion.

  7. The unique organization of filamentous actin in the medullary canal of the medulla oblongata.

    PubMed

    Tan, Bai-Hong; Guo, Chun-Yan; Xiong, Tian-Qing; Chen, Ling-Meng; Li, Yan-Chao

    2017-01-24

    In the central canal, F-actin is predominantly localized in the apical region, forming a ring-like structure around the circumference of the lumen. However, an exception is found in the medulla oblongata, where the apical F-actin becomes interrupted in the ventral aspect of the canal. To clarify the precise localization of F-actin, the fluorescence signals for F-actin were converted to the peroxidase/DAB reaction products in this study by a phalloidin-based ultrastructural technique, which demonstrated that F-actin is located mainly in the microvilli and terminal webs in the ependymocytes. It is because the ventrally oriented ependymocytes do not possess well-developed microvilli or terminal web that led to a discontinuous labeling of F-actin in the medullary canal. Since spinal motions can change the shape and size of the central canal, we next examined the cytoskeletons in the medullary canal in both rats and monkeys, because these two kinds of animals show different kinematics at the atlanto-occipital articulation. Our results first demonstrated that the apical F-actin in the medullary canal is differently organized in the animals with different head-neck kinemics, which suggests that the mechanic stretching of spinal motions is capable of inducing F-actin reorganization and the subsequent cell-shape changes in the central canal.

  8. Using cell structures to develop functional nanomaterials and nanostructures--case studies of actin filaments and microtubules.

    PubMed

    Wu, Kevin Chia-Wen; Yang, Chung-Yao; Cheng, Chao-Min

    2014-04-25

    This article is based on the continued development of biologically relevant elements (i.e., actin filaments and microtubules in living cells) as building blocks to create functional nanomaterials and nanostructures that can then be used to manufacture nature-inspired small-scale devices or systems. Here, we summarize current progress in the field and focus specifically on processes characterized by (1) robustness and ease of use, (2) inexpensiveness, and (3) potential expandability to mass production. This article, we believe, will provide scientists and engineers with a more comprehensive understanding of how to mine biological materials and natural design features to construct functional materials and devices.

  9. ForC lacks canonical formin activity but bundles actin filaments and is required for multicellular development of Dictyostelium cells.

    PubMed

    Junemann, Alexander; Winterhoff, Moritz; Nordholz, Benjamin; Rottner, Klemens; Eichinger, Ludwig; Gräf, Ralph; Faix, Jan

    2013-01-01

    Diaphanous-related formins (DRFs) drive the nucleation and elongation of linear actin filaments downstream of Rho GTPase signalling pathways. Dictyostelium formin C (ForC) resembles a DRF, except that it lacks a genuine formin homology domain 1 (FH1), raising the questions whether or not ForC can nucleate and elongate actin filaments. We found that a recombinant ForC-FH2 fragment does not nucleate actin polymerization, but moderately decreases the rate of spontaneous actin assembly and disassembly, although the barbed-end elongation rate in the presence of the formin was not markedly changed. However, the protein bound to and crosslinked actin filaments into loose bundles of mixed polarity. Furthermore, ForC is an important regulator of morphogenesis since ForC-null cells are severely impaired in development resulting in the formation of aberrant fruiting bodies. Immunoblotting revealed that ForC is absent during growth, but becomes detectable at the onset of early aggregation when cells chemotactically stream together to form a multicellular organism, and peaks around the culmination stage. Fluorescence microscopy of cells ectopically expressing a GFP-tagged, N-terminal ForC fragment showed its prominent accumulation in the leading edge, suggesting that ForC may play a role in cell migration. In agreement with its expression profile, no defects were observed in random migration of vegetative mutant cells. Notably, chemotaxis of starved cells towards a source of cAMP was severely impaired as opposed to control. This was, however, largely due to a marked developmental delay of the mutant, as evidenced by the expression profile of the early developmental marker csA. In line with this, chemotaxis was almost restored to wild type levels after prolonged starvation. Finally, we observed a complete failure of phototaxis due to abolished slug formation and a massive reduction of spores consistent with forC promoter-driven expression of β-galactosidase in prespore cells

  10. As functional nuclear actin comes into view, is it globular, filamentous, or both?

    PubMed Central

    Pederson, Thoru

    2008-01-01

    The idea that actin may have an important function in the nucleus has undergone a rapid transition from one greeted with skepticism to a now rapidly advancing research field. Actin has now been implicated in transcription by all three RNA polymerases, but the structural form it adopts in these processes remains unclear. Recently, a claim was made that monomeric nuclear actin plays a role in signal transduction, while a just-published study of RNA polymerase I transcription has implicated polymeric actin, consorting with an isoform of its classical partner myosin. Both studies are critically discussed here, and although there are several issues to be resolved, it now seems reasonable to start thinking about functions for both monomeric and assembled actin in the nucleus. PMID:18347069

  11. The effect of thin filament activation on the attachment of weak binding cross-bridges: A two-dimensional x-ray diffraction study on single muscle fibers.

    PubMed

    Kraft, T; Xu, S; Brenner, B; Yu, L C

    1999-03-01

    To study possible structural changes in weak cross-bridge attachment to actin upon activation of the thin filament, two-dimensional (2D) x-ray diffraction patterns of skinned fibers from rabbit psoas muscle were recorded at low and high calcium concentration in the presence of saturating concentrations of MgATPgammaS, a nucleotide analog for weak binding states. We also studied 2D x-ray diffraction patterns recorded under relaxing conditions at an ionic strength above and below 50 mM, because it had been proposed from solution studies that reducing ionic strength below 50 mM also induces activation of the thin filament. For this project a novel preparation had to be established that allows recording of 2D x-ray diffraction patterns from single muscle fibers instead of natural fiber bundles. This was required to minimize substrate depletion or product accumulation within the fibers. When the calcium concentration was raised, the diffraction patterns recorded with MgATPgammaS revealed small changes in meridional reflections and layer line intensities that could be attributed in part to the effects of calcium binding to the thin filament (increase in I380, decrease in first actin layer line intensity, increase in I59) and in part to small structural changes of weakly attached cross-bridges (e.g., increase in I143 and I72). Calcium-induced small-scale structural rearrangements of cross-bridges weakly attached to actin in the presence of MgATPgammaS are consistent with our previous observation of reduced rate constants for attachment and detachment of cross-bridges with MgATPgammaS at high calcium. Yet, no evidence was found that weakly attached cross-bridges change their mode of attachment toward a stereospecific conformation when the actin filament is activated by adding calcium. Similarly, reducing ionic strength to less than 50 mM does not induce a transition from nonstereospecific to stereospecific attachment.

  12. Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana

    PubMed Central

    2016-01-01

    Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements. PMID:27310016

  13. Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana.

    PubMed

    Suetsugu, Noriyuki; Higa, Takeshi; Gotoh, Eiji; Wada, Masamitsu

    2016-01-01

    Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements.

  14. Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction

    SciTech Connect

    Sugimoto, Yasunobu Takezawa, Yasunori; Matsuo, Tatsuhito; Ueno, Yutaka; Minakata, Shiho; Tanaka, Hidehiro; Wakabayashi, Katsuzo

    2008-04-25

    In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4 nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by {approx}0.1% upon activation relative to the relaxing state and increased by {approx}0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca{sup 2+}-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca{sup 2+}-binding and the second induced by actomyosin interaction.

  15. Thin filament diversity and physiological properties of fast and slow fiber types in astronaut leg muscles

    NASA Technical Reports Server (NTRS)

    Riley, Danny A.; Bain, James L W.; Thompson, Joyce L.; Fitts, Robert H.; Widrick, Jeffrey J.; Trappe, Scott W.; Trappe, Todd A.; Costill, David L.

    2002-01-01

    Slow type I fibers in soleus and fast white (IIa/IIx, IIx), fast red (IIa), and slow red (I) fibers in gastrocnemius were examined electron microscopically and physiologically from pre- and postflight biopsies of four astronauts from the 17-day, Life and Microgravity Sciences Spacelab Shuttle Transport System-78 mission. At 2.5-microm sarcomere length, thick filament density is approximately 1,012 filaments/microm(2) in all fiber types and unchanged by spaceflight. In preflight aldehyde-fixed biopsies, gastrocnemius fibers possess higher percentages (approximately 23%) of short thin filaments than soleus (9%). In type I fibers, spaceflight increases short, thin filament content from 9 to 24% in soleus and from 26 to 31% in gastrocnemius. Thick and thin filament spacing is wider at short sarcomere lengths. The Z-band lattice is also expanded, except for soleus type I fibers with presumably stiffer Z bands. Thin filament packing density correlates directly with specific tension for gastrocnemius fibers but not soleus. Thin filament density is inversely related to shortening velocity in all fibers. Thin filament structural variation contributes to the functional diversity of normal and spaceflight-unloaded muscles.

  16. Observation of Actin Filaments in Leydig Cells with a Contact-type Soft X-ray Microscope with Laser Plasma X-ray Source

    NASA Astrophysics Data System (ADS)

    Kado, Masataka; Ishino, Masahiko; Tamotsu, Satoshi; Yasuda, Keiko; Kishimoto, Maki; Nishikino, Masaharu; Kinjo, Yasuhito; Shinohara, Kunio

    Actin filaments in Leydig cells from mouse testes have been observed with a contact-type soft x-ray microscope with laser plasma x-ray source. The Leydig cells were fixed with paraformaldehyde, stained with Phalloidin, and observed with a confocal laser microscope prior to the observation with x-ray microscope. Obtained images by both of the confocal laser microscopy and the x-ray microscopy were directly compared and revealed that not only position of actin filaments but also the shapes can be identified each other. The actin filaments in the x-ray images were clearly recognized and their structures were obtained in more detail compared to those in the confocal laser microscope images.

  17. Eukaryotic cell locomotion depends on the propagation of self-organized reaction-diffusion waves and oscillations of actin filament assembly.

    PubMed

    Vicker, Michael G

    2002-04-15

    Actin filament (F-actin) assembly kinetics determines the locomotion and shape of crawling eukaryotic cells, but the nature of these kinetics and their determining reactions are unclear. Live BHK21 fibroblasts, mouse melanoma cells, and Dictyostelium amoebae, locomoting on glass and expressing Green Fluorescent Protein-actin fusion proteins, were examined by confocal microscopy. The cells demonstrated three-dimensional bands of F-actin, which propagated throughout the cytoplasm at rates usually ranging between 2 and 5 microm/min in each cell type and produced lamellipodia or pseudopodia at the cell boundary. F-actin's dynamic behavior and supramolecular spatial patterns resembled in detail self-organized chemical waves in dissipative, physico-chemical systems. On this basis, the present observations provide the first evidence of self-organized, and probably autocatalytic, chemical reaction-diffusion waves of reversible actin filament assembly in vertebrate cells and a comprehensive record of wave and locomotory dynamics in vegetative-stage Dictyostelium cells. The intensity and frequency of F-actin wavefronts determine locomotory cell projections and the rotating oscillatory waves, which structure the cell surface. F-actin assembly waves thus provide a fundamental, deterministic, and nonlinear mechanism of cell locomotion and shape, which complements mechanisms based exclusively on stochastic molecular reaction kinetics.

  18. Simulated ischaemia induces Ca2+-independent glutamatergic vesicle release through actin filament depolymerization in area CA1 of the hippocampus.

    PubMed

    Andrade, Adriana L; Rossi, David J

    2010-05-01

    Transient, non-catastrophic brain ischaemia can induce either a protected state against subsequent episodes of ischaemia (ischaemic preconditioning) or delayed, selective neuronal death. Altered glutamatergic signalling and altered Ca(2+) homeostasis have been implicated in both processes. Here we use simultaneous patch-clamp recording and Ca(2+) imaging to monitor early changes in glutamate release and cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in an in vitro slice model of hippocampal ischaemia. In slices loaded with the Ca(2+)-sensitive dye Fura-2, ischaemia leads to an early increase in [Ca(2+)](c) that precedes the severe ischaemic depolarization (ID) associated with pan necrosis. The early increase in [Ca(2+)](c) is mediated by influx through the plasma membrane and release from internal stores, and parallels an early increase in vesicular glutamate release that manifests as a fourfold increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). However, the increase in mEPSC frequency is not prevented by blocking the increase in [Ca(2+)](c), and the early rise in [Ca(2+)](c) is not affected by blocking ionotropic and metabotropic glutamate receptors. Thus, the increase in [Ca(2+)](c) and the increase in glutamate release are independent of each other. Stabilizing actin filaments with jaspamide or phalloidin prevented vesicle release induced by ischaemia. Our results identify several early cellular cascades triggered by ischaemia: Ca(2+) influx, Ca(2+) release from intracellular stores, actin filament depolymerization, and vesicular release of glutamate that depends on actin dynamics but not [Ca(2+)](c). All of these processes precede the catastrophic ID by several minutes, and thus represent potential target mechanisms to influence the outcome of an ischaemic episode.

  19. Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells.

    PubMed

    Yokota, Etsuo; Vidali, Luis; Tominaga, Motoki; Tahara, Hiroshi; Orii, Hidefumi; Morizane, Yosuke; Hepler, Peter K; Shimmen, Teruo

    2003-10-01

    In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin

  20. The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis.

    PubMed

    Henson, John H; Ditzler, Casey E; Germain, Aphnie; Irwin, Patrick M; Vogt, Eric T; Yang, Shucheng; Wu, Xufeng; Shuster, Charles B

    2017-03-01

    Despite recent advances in our understanding of the components and spatial regulation of the contractile ring (CR), the precise ultrastructure of actin and myosin II within the animal cell CR remains an unanswered question. We used superresolution light microscopy and platinum replica transmission electron microscopy (TEM) to determine the structural organization of actin and myosin II in isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination microscopy indicated that within the CR, actin and myosin II filaments were organized into tightly packed linear arrays oriented along the axis of constriction and restricted to a narrow zone within the furrow. In contrast, myosin II filaments in earlier stages of cytokinesis were organized into small, discrete, and regularly spaced clusters. TEM showed that actin within the CR formed a dense and anisotropic array of elongate, antiparallel filaments, whereas myosin II was organized into laterally associated, head-to-head filament chains highly reminiscent of mammalian cell stress fibers. Together these results not only support the canonical "purse-string" model for contractile ring constriction, but also suggest that the CR may be derived from foci of myosin II filaments in a manner similar to what has been demonstrated in fission yeast.

  1. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front

    PubMed Central

    Nguyen, Trang Thi Thu; Park, Wei Sun; Park, Byung Ouk; Kim, Cha Yeon; Oh, Yohan; Kim, Jin Man; Choi, Hana; Kyung, Taeyoon; Kim, Cheol-Hee; Lee, Gabsang; Hahn, Klaus M.; Meyer, Tobias; Heo, Won Do

    2016-01-01

    Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration. PMID:27555588

  2. Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile, long-lived plaques.

    PubMed

    Meijer, Harold J G; Hua, Chenlei; Kots, Kiki; Ketelaar, Tijs; Govers, Francine

    2014-06-01

    The actin cytoskeleton is a dynamic but well-organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact-eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub-apical region whereas in the extreme apex in growing hyphae, waves of fine F-actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ∼30 s while disassembly was accomplished in ∼10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.

  3. Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay.

    PubMed

    Debold, Edward P; Turner, Matthew A; Stout, Jordan C; Walcott, Sam

    2011-06-01

    Elevated levels of inorganic phosphate (P(i)) are believed to inhibit muscular force by reversing myosin's force-generating step. These same levels of P(i) can also affect muscle velocity, but the molecular basis underlying these effects remains unclear. We directly examined the effect of P(i) (30 mM) on skeletal muscle myosin's ability to translocate actin (V(actin)) in an in vitro motility assay. Manipulation of the pH enabled us to probe rebinding of P(i) to myosin's ADP-bound state, while changing the ATP concentration probed rebinding to the rigor state. Surprisingly, the addition of P(i) significantly increased V(actin) at both pH 6.8 and 6.5, causing a doubling of V(actin) at pH 6.5. To probe the mechanisms underlying this increase in speed, we repeated these experiments while varying the ATP concentration. At pH 7.4, the effects of P(i) were highly ATP dependent, with P(i) slowing V(actin) at low ATP (<500 μM), but with a minor increase at 2 mM ATP. The P(i)-induced slowing of V(actin), evident at low ATP (pH 7.4), was minimized at pH 6.8 and completely reversed at pH 6.5. These data were accurately fit with a simple detachment-limited kinetic model of motility that incorporated a P(i)-induced prolongation of the rigor state, which accounted for the slowing of V(actin) at low ATP, and a P(i)-induced detachment from a strongly bound post-power-stroke state, which accounted for the increase in V(actin) at high ATP. These findings suggest that P(i) differentially affects myosin function: enhancing velocity, if it rebinds to the ADP-bound state, while slowing velocity, if it binds to the rigor state.

  4. Disproportionate loss of thin filaments in human soleus muscle after 17-day bed rest

    NASA Technical Reports Server (NTRS)

    Riley, D. A.; Bain, J. L.; Thompson, J. L.; Fitts, R. H.; Widrick, J. J.; Trappe, S. W.; Trappe, T. A.; Costill, D. L.

    1998-01-01

    Previously we reported that, after 17-day bed rest unloading of 8 humans, soleus slow fibers atrophied and exhibited increased velocity of shortening without fast myosin expression. The present ultrastructural study examined fibers from the same muscle biopsies to determine whether decreased myofilament packing density accounted for the observed speeding. Quantitation was by computer-assisted morphometry of electron micrographs. Filament densities were normalized for sarcomere length, because density depends directly on length. Thick filament density was unchanged by bed rest. Thin filaments/microm2 decreased 16-23%. Glycogen filled the I band sites vacated by filaments. The percentage decrease in thin filaments (Y) correlated significantly (P < 0.05) with the percentage increase in velocity (X), (Y = 0.1X + 20%, R2 = 0.62). An interpretation is that fewer filaments increases thick to thin filament spacing and causes earlier cross-bridge detachment and faster cycling. Increased velocity helps maintain power (force x velocity) as atrophy lowers force. Atrophic muscles may be prone to sarcomere reloading damage because force/microm2 was near normal, and force per thin filament increased an estimated 30%.

  5. A 45,000-mol-wt protein from unfertilized sea urchin eggs severs actin filaments in a calcium-dependent manner and increases the steady-state concentration of nonfilamentous actin

    PubMed Central

    1984-01-01

    A 45,000-mol-wt protein has been purified from unfertilized sea urchin (Strongylocentrotus purpuratus) eggs. The isolation scheme includes DEAE cellulose ion-exchange chromatography, gel filtration, and hydroxylapatite chromatography. The homogeneity of the isolated protein is greater than 90% by SDS PAGE. The 45,000-mol-wt protein reduces the viscosity of actin filaments in a Ca2+-dependent manner. The free calcium concentration required for the activity of this protein is in the micromolar range. Electron microscopic studies reveal that the formation of short filaments parallels the decrease in viscosity. Energy transfer and sedimentation experiments indicate a net disassembly of actin filaments and an increase in the steady-state nonfilamentous actin concentration in the presence of Ca2+ ions and the 45,000-mol-wt protein. The increase in the steady-state nonfilamentous actin concentration is proportional to the amount of 45,000-mol-wt protein added. The actin molecules disassembled by the addition of the 45,000-mol-wt protein are capable of polymerization. PMID:6540784

  6. A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

    PubMed Central

    Horsington, Jacquelyn; Lynn, Helena; Turnbull, Lynne; Cheng, Delfine; Braet, Filip; Diefenbach, Russell J.; Whitchurch, Cynthia B.; Karupiah, Guna; Newsome, Timothy P.

    2013-01-01

    Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5P189S mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin

  7. Trimers of the fibronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation.

    PubMed

    Coussen, Françoise; Choquet, Daniel; Sheetz, Michael P; Erickson, Harold P

    2002-06-15

    Previous studies have shown that small beads coated with FN7-10, a four-domain cell adhesion fragment of fibronectin, bind to cell surfaces and translocate rearward. Here we investigate whether soluble constructs containing two to five FN7-10 units might be sufficient for activity. We have produced a monomer, three forms of dimers, a trimer and a pentamer of FN7-10, on the end of spacer arms. These oligomers could bind small clusters of up to five integrins. Fluorescence microscopy showed that the trimer and pentamer bound strongly to the cell surface, and within 5 minutes were prominently localized to actin fiber bundles. Monomers and dimers showed only diffuse localization. Beads coated with a low concentration (probably one complex per bead) of trimer or pentamer showed prolonged binding and rearward translocation, presumably with the translocating actin cytskeleton. Beads containing monomer or dimer showed only brief binding and diffusive movements. We conclude that clusters of three integrin-binding ligands are necessary and sufficient for coupling to and translocating with the actin cytoskeleton.

  8. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    SciTech Connect

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  9. Skeletal muscle fiber atrophy: altered thin filament density changes slow fiber force and shortening velocity.

    PubMed

    Riley, D A; Bain, J L W; Romatowski, J G; Fitts, R H

    2005-02-01

    Single skinned fibers from soleus and adductor longus (AL) muscles of weight-bearing control rats and rats after 14-day hindlimb suspension unloading (HSU) were studied physiologically and ultrastructurally to investigate how slow fibers increase shortening velocity (V0) without fast myosin. We hypothesized that unloading and shortening of soleus during HSU reduces densities of thin filaments, generating wider myofilament separations that increase V0 and decrease specific tension (kN/m2). During HSU, plantarflexion shortened soleus working length 23%. AL length was unchanged. Both muscles atrophied as shown by reductions in fiber cross-sectional area. For AL, the 60% atrophy accounted fully for the 58% decrease in absolute tension (mN). In the soleus, the 67% decline in absolute tension resulted from 58% atrophy plus a 17% reduction in specific tension. Soleus fibers exhibited a 25% reduction in thin filaments, whereas there was no change in AL thin filament density. Loss of thin filaments is consistent with reduced cross bridge formation, explaining the fall in specific tension. V0 increased 27% in soleus but was unchanged in AL. The V0 of control and HSU fibers was inversely correlated (R = -0.83) with thin filament density and directly correlated (R = 0.78) with thick-to-thin filament spacing distance in a nonlinear fashion. These data indicate that reduction in thin filament density contributes to an increased V0 in slow fibers. Osmotically compacting myofilaments with 5% dextran returned density, spacing, and specific tension and slowed V0 to near-control levels and provided evidence for myofilament spacing modulating tension and V0.

  10. RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes

    PubMed Central

    Wang, Yu; Liu, Ou; Zhang, Jing; Gleason, Adenrele; Yang, Zhenrong; Wang, Hui; Shi, Anbing; Grant, Barth D.

    2016-01-01

    EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation. PMID:27272733

  11. Actin, microvilli, and the fertilization cone of sea urchin eggs

    PubMed Central

    1980-01-01

    Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges. PMID:6893988

  12. WAVE2, N-WASP, and Mena facilitate cell invasion via phosphatidylinositol 3-kinase-dependent local accumulation of actin filaments.

    PubMed

    Takahashi, Kazuhide; Suzuki, Katsuo

    2011-11-01

    Cell migration is accomplished by the formation of cellular protrusions such as lamellipodia and filopodia. These protrusions result from actin filament (F-actin) rearrangement at the cell cortex by WASP/WAVE family proteins and Drosophila enabled (Ena)/vasodilator-stimulated factor proteins. However, the role of each of these actin cytoskeletal regulatory proteins in the regulation of three-dimensional cell invasion remains to be clarified. We found that platelet-derived growth factor (PDGF) induces invasion of MDA-MB-231 human breast cancer cells through invasion chamber membrane pores. This invasion was accompanied by intensive F-actin accumulation at the sites of cell infiltration. After PDGF stimulation, WAVE2, N-WASP, and a mammalian Ena (Mena) colocalized with F-actin at the sites of cell infiltration in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Depletion of WAVE2, N-WASP, or Mena by RNA interference (RNAi) abrogated both cell invasion and intensive F-actin accumulation at the invasion site. These results indicate that by mediating intensive F-actin accumulation at the sites of cell infiltration, WAVE2, N-WASP, and Mena are crucial for PI3K-dependent cell invasion induced by PDGF.

  13. C-terminal fragment of amebin promotes actin filament bundling, inhibits acto-myosin ATPase activity and is essential for amoeba migration.

    PubMed

    Jóźwiak, Jolanta; Rzhepetskyy, Yuriy; Sobczak, Magdalena; Kocik, Elżbieta; Skórzewski, Radosław; Kłopocka, Wanda; Rędowicz, Maria Jolanta

    2011-02-01

    Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca(2+)-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.

  14. Contributions of Ca2+-Independent Thin Filament Activation to Cardiac Muscle Function

    PubMed Central

    Aboelkassem, Yasser; Bonilla, Jordan A.; McCabe, Kimberly J.; Campbell, Stuart G.

    2015-01-01

    Although Ca2+ is the principal regulator of contraction in striated muscle, in vitro evidence suggests that some actin-myosin interaction is still possible even in its absence. Whether this Ca2+-independent activation (CIA) occurs under physiological conditions remains unclear, as does its potential impact on the function of intact cardiac muscle. The purpose of this study was to investigate CIA using computational analysis. We added a structurally motivated representation of this phenomenon to an existing myofilament model, which allowed predictions of CIA-dependent muscle behavior. We found that a certain amount of CIA was essential for the model to reproduce reported effects of nonfunctional troponin C on myofilament force generation. Consequently, those data enabled estimation of ΔGCIA, the energy barrier for activating a thin filament regulatory unit in the absence of Ca2+. Using this estimate of ΔGCIA as a point of reference (∼7 kJ mol−1), we examined its impact on various aspects of muscle function through additional simulations. CIA decreased the Hill coefficient of steady-state force while increasing myofilament Ca2+ sensitivity. At the same time, CIA had minimal effect on the rate of force redevelopment after slack/restretch. Simulations of twitch tension show that the presence of CIA increases peak tension while profoundly delaying relaxation. We tested the model’s ability to represent perturbations to the Ca2+ regulatory mechanism by analyzing twitch records measured in transgenic mice expressing a cardiac troponin I mutation (R145G). The effects of the mutation on twitch dynamics were fully reproduced by a single parameter change, namely lowering ΔGCIA by 2.3 kJ mol−1 relative to its wild-type value. Our analyses suggest that CIA is present in cardiac muscle under normal conditions and that its modulation by gene mutations or other factors can alter both systolic and diastolic function. PMID:26588569

  15. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

    PubMed

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

  16. Ca(2+)- and S1-induced movement of troponin T on reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy.

    PubMed

    Kimura, Chieko; Maeda, Kayo; Maéda, Yuichiro; Miki, Masao

    2002-07-01

    Troponin T (TnT) is an essential component of troponin (Tn) for the Ca(2+)-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH(2)-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal beta-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca(2+) binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 A, respectively in the absence of Ca(2+), and increased by 2.6 and 5.8 A, respectively upon binding of Ca(2+) to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 A respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca(2+)- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca(2+), the rigor S1 binding also increased the distances to the same extent as the presence of Ca(2+) when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca(2+). However, plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca(2+) but sigmoidal curve in the absence of Ca(2+). FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET

  17. Very Fast Temperature Measurement with a Thin Lamp Filament

    ERIC Educational Resources Information Center

    G. Calza, Gratton, L. M.; Lopez-Arias, T.; Oss, S.

    2012-01-01

    We construct a thermometer exploiting the electric resistance of the filament of a small lamp used in micro-illumination settings. The instrument may guarantee a response time better than 10 ms, i.e. much faster than commercial thermocouples or other quite expensive devices. This makes our thermometer a useful one in several processes which are…

  18. Role of Actin Filaments in Correlating Nuclear Shape and Cell Spreading

    PubMed Central

    Vishavkarma, Renu; Raghavan, Swetavalli; Kuyyamudi, Chandrashekar; Majumder, Abhijit; Dhawan, Jyotsna; Pullarkat, Pramod A.

    2014-01-01

    It is well known that substrate properties like stiffness and adhesivity influence stem cell morphology and differentiation. Recent experiments show that cell morphology influences nuclear geometry and hence gene expression profile. The mechanism by which surface properties regulate cell and nuclear properties is only beginning to be understood. Direct transmission of forces as well as chemical signalling are involved in this process. Here, we investigate the formal aspect by studying the correlation between cell spreading and nuclear deformation using Mesenchymal stem cells under a wide variety of conditions. It is observed that a robust quantitative relation holds between the cell and nuclear projected areas, irrespective of how the cell area is modified or when various cytoskeletal or nuclear components are perturbed. By studying the role of actin stress fibers in compressing the nucleus we propose that nuclear compression by stress fibers can lead to enhanced cell spreading due to an interplay between elastic and adhesion factors. The significance of myosin-II in regulating this process is also explored. We demonstrate this effect using a simple technique to apply external compressive loads on the nucleus. PMID:25251154

  19. The effect of thin filament activation on the attachment of weak binding cross-bridges: A two-dimensional x-ray diffraction study on single muscle fibers.

    PubMed Central

    Kraft, T; Xu, S; Brenner, B; Yu, L C

    1999-01-01

    To study possible structural changes in weak cross-bridge attachment to actin upon activation of the thin filament, two-dimensional (2D) x-ray diffraction patterns of skinned fibers from rabbit psoas muscle were recorded at low and high calcium concentration in the presence of saturating concentrations of MgATPgammaS, a nucleotide analog for weak binding states. We also studied 2D x-ray diffraction patterns recorded under relaxing conditions at an ionic strength above and below 50 mM, because it had been proposed from solution studies that reducing ionic strength below 50 mM also induces activation of the thin filament. For this project a novel preparation had to be established that allows recording of 2D x-ray diffraction patterns from single muscle fibers instead of natural fiber bundles. This was required to minimize substrate depletion or product accumulation within the fibers. When the calcium concentration was raised, the diffraction patterns recorded with MgATPgammaS revealed small changes in meridional reflections and layer line intensities that could be attributed in part to the effects of calcium binding to the thin filament (increase in I380, decrease in first actin layer line intensity, increase in I59) and in part to small structural changes of weakly attached cross-bridges (e.g., increase in I143 and I72). Calcium-induced small-scale structural rearrangements of cross-bridges weakly attached to actin in the presence of MgATPgammaS are consistent with our previous observation of reduced rate constants for attachment and detachment of cross-bridges with MgATPgammaS at high calcium. Yet, no evidence was found that weakly attached cross-bridges change their mode of attachment toward a stereospecific conformation when the actin filament is activated by adding calcium. Similarly, reducing ionic strength to less than 50 mM does not induce a transition from nonstereospecific to stereospecific attachment. PMID:10049330

  20. Pre-eruption Oscillations in Thin and Long Features in a Quiescent Filament

    NASA Astrophysics Data System (ADS)

    Joshi, Anand D.; Hanaoka, Yoichiro; Suematsu, Yoshinori; Morita, Satoshi; Yurchyshyn, Vasyl; Cho, Kyung-Suk

    2016-12-01

    We investigate the eruption of a quiescent filament located close to an active region. Large-scale activation was observed in only half of the filament in the form of pre-eruption oscillations. Consequently only this half erupted nearly 30 hr after the oscillations commenced. Time-slice diagrams of 171 Å images from the Atmospheric Imaging Assembly were used to study the oscillations. These were observed in several thin and long features connecting the filament spine to the chromosphere below. This study traces the origin of such features and proposes their possible interpretation. Small-scale magnetic flux cancellation accompanied by a brightening was observed at the footpoint of the features shortly before their appearance, in images recorded by the Helioseismic and Magnetic Imager. A slow rise of the filament was detected in addition to the oscillations, indicating a gradual loss of equilibrium. Our analysis indicates that a change in magnetic field connectivity between two neighbouring active regions and the quiescent filament resulted in a weakening of the overlying arcade of the filament, leading to its eruption. It is also suggested that the oscillating features are filament barbs, and the oscillations are a manifestation during the pre-eruption phase of the filaments.

  1. Mammalian class I myosin, Myo1b, is monomeric and cross-links actin filaments as determined by hydrodynamic studies and electron microscopy.

    PubMed

    Stafford, Walter F; Walker, Matt L; Trinick, John A; Coluccio, Lynne M

    2005-01-01

    The class I myosin, Myo1b, is a calmodulin- and actin-associated molecular motor widely expressed in mammalian tissues. Analytical ultracentrifugation studies indicate that Myo1b purified from rat liver has a Stokes radius of 6.7 nm and a sedimentation coefficient, s(20,w), of 7.0 S with a predicted molar mass of 213 kg/mol. These results indicate that Myo1b is monomeric and consists primarily of a splice variant having five associated calmodulins. Molecular modeling based on the analytical ultracentrifugation studies are supported by electron microscopy studies that depict Myo1b as a single-headed, tadpole-shaped molecule with outer dimensions of 27.9 x 4.0 nm. Above a certain Myo1b/actin ratio, Myo1b bundles actin filaments presumably by virtue of a second actin-binding site. These studies provide new information regarding the oligomeric state and morphology of Myo1b and support a model in which Myo1b cross-links actin through a cryptic actin-binding site.

  2. Viscoelastic dynamics of actin filaments coupled to rotary F-ATPase: angular torque profile of the enzyme.

    PubMed Central

    Pänke, O; Cherepanov, D A; Gumbiowski, K; Engelbrecht, S; Junge, W

    2001-01-01

    ATP synthase (F(O)F(1)) operates as two rotary motor/generators coupled by a common shaft. Both portions, F(1) and F(O), are rotary steppers. Their symmetries are mismatched (C(3) versus C(10-14)). We used the curvature of fluorescent actin filaments, attached to the rotating c-ring, as a spring balance (flexural rigidity of 8. 10(-26) Nm(2)) to gauge the angular profile of the output torque at F(O) during ATP hydrolysis by F(1) (see theoretical companion article (. Biophys. J. 81:1234-1244.)). The large average output torque (50 +/- 6 pN. nm) proved the absence of any slip. Variations of the torque were small, and the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the threefold stepping and high activation barrier of the driving motor proper, the rather constant output torque implied a soft elastic power transmission between F(1) and F(O). It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate of the two counteracting and stepping motor/generators. PMID:11509339

  3. Disruption of actin filaments and suppression of pancreatic cancer cell viability and migration following treatment with polyisoprenylated cysteinyl amides

    PubMed Central

    Nkembo, Augustine T; Salako, Olufisayo; Poku, Rosemary A; Amissah, Felix; Ntantie, Elizabeth; Flores-Rozas, Hernan; Lamango, Nazarius S

    2016-01-01

    Pancreatic cancer is characterized by K-Ras mutations in over 90% of the cases. The mutations make the tumors aggressive and resistant to current therapies resulting in very poor prognoses. Valiant efforts to drug mutant K-Ras and related proteins for the treatment of cancers with Ras mutations have been elusive. The need thus persists for therapies to target and suppress the hyperactive K-Ras mutant proteins to normal levels of activity. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) of polyisoprenylated methylated protein methyl esterase (PMPMEase) were designed to disrupt polyisoprenylated protein metabolism and/or functions. The potential for PCAIs to serve as targeted anticancer agents for pancreatic cancer was evaluated in pancreatic ductal adenocarcinoma (PDAC) cell lines expressing mutant (MIAPaCa-2 and Panc-1) and wild type (BxPC-3) K-Ras proteins. The PCAIs inhibited MIAPaCa-2 and BxPC-3 cell viability and induced apoptosis with EC50 values as low as 1.9 µM. The PCAIs, at 0.5 µM, inhibited MIAPaCa-2 cell migration by 50%, inhibited colony formation and disrupted F-actin filament organization. The PCAIs blocked MIAPaCa-2 cell progression at the G0/G1 phase. These results reveal that the PCAIs disrupt pertinent biological processes that lead to pancreatic cancer progression and thus have the potential to act as targeted effective treatments for pancreatic cancer. PMID:27904769

  4. Septin 9_i2 is downregulated in tumors, impairs cancer cell migration and alters subnuclear actin filaments

    PubMed Central

    Verdier-Pinard, P.; Salaun, D.; Bouguenina, H.; Shimada, S.; Pophillat, M.; Audebert, S.; Agavnian, E.; Coslet, S.; Charafe-Jauffret, E.; Tachibana, T.; Badache, A.

    2017-01-01

    Functions of septin cytoskeletal polymers in tumorigenesis are still poorly defined. Their role in the regulation of cytokinesis and cell migration were proposed to contribute to cancer associated aneuploidy and metastasis. Overexpression of Septin 9 (Sept9) promotes migration of cancer cell lines. SEPT9 mRNA and protein expression is increased in breast tumors compared to normal and peritumoral tissues and amplification of SEPT9 gene was positively correlated with breast tumor progression. However, the existence of multiple isoforms of Sept9 is a confounding factor in the analysis of Sept9 functions. In the present study, we analyze the protein expression of Sept9_i2, an uncharacterized isoform, in breast cancer cell lines and tumors and describe its specific impact on cancer cell migration and Sept9 cytoskeletal distribution. Collectively, our results showed that, contrary to Sept9_i1, Sept9_i2 did not support cancer cell migration, and induced a loss of subnuclear actin filaments. These effects were dependent on Sept9_i2 specific N-terminal sequence. Sept9_i2 was strongly down-regulated in breast tumors compared to normal mammary tissues. Thus our data indicate that Sept9_i2 is a negative regulator of breast tumorigenesis. We propose that Sept9 tumorigenic properties depend on the balance between Sept9_i1 and Sept9_i2 expression levels. PMID:28338090

  5. A model of filament-wound thin cylinders

    NASA Technical Reports Server (NTRS)

    Calius, Emilio P.; Springer, George S.

    1990-01-01

    A model was developed for simulating he manufacturing process of filament-wound cylinders made of a thermoset matrix composite. The model relates the process variables (winding speed, fiber tension, applied temperature) to the parameters characterizing the composite cylinder and the mandrel. The model is applicable to cylinders for which the diameter is large compared to the wall thickness. The model was implemented by a user-friendly computer code suitable for generating numerical results.

  6. Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle

    PubMed Central

    Zhang, Xuemeng; Kampourakis, Thomas; Yan, Ziqian; Sevrieva, Ivanka; Irving, Malcolm; Sun, Yin-Biao

    2017-01-01

    The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures the volume of blood ejected in each heartbeat is matched to the extent of venous filling. At the cellular level, heart muscle cells generate higher force when stretched, but despite intense efforts the underlying molecular mechanism remains unknown. We applied a fluorescence-based method, which reports structural changes separately in the thick and thin filaments of rat cardiac muscle, to elucidate that mechanism. The distinct structural changes of troponin C in the thin filaments and myosin regulatory light chain in the thick filaments allowed us to identify two aspects of the Frank-Starling relation. Our results show that the enhanced force observed when heart muscle cells are maximally activated by calcium is due to a change in thick filament structure, but the increase in calcium sensitivity at lower calcium levels is due to a change in thin filament structure. DOI: http://dx.doi.org/10.7554/eLife.24081.001 PMID:28229860

  7. Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression.

    PubMed

    Haynes, Jennifer; Srivastava, Jyoti; Madson, Nikki; Wittmann, Torsten; Barber, Diane L

    2011-12-01

    Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.

  8. αT-Catenin Is a Constitutive Actin-binding α-Catenin That Directly Couples the Cadherin·Catenin Complex to Actin Filaments*

    PubMed Central

    Wickline, Emily D.; Dale, Ian W.; Merkel, Chelsea D.; Heier, Jonathon A.; Stolz, Donna B.

    2016-01-01

    α-Catenin is the primary link between the cadherin·catenin complex and the actin cytoskeleton. Mammalian αE-catenin is allosterically regulated: the monomer binds the β-catenin·cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin. As part of the cadherin·catenin complex, αE-catenin requires force to bind F-actin strongly. It is not known whether these properties are conserved across the mammalian α-catenin family. Here we show that αT (testes)-catenin, a protein unique to amniotes that is expressed predominantly in the heart, is a constitutive actin-binding α-catenin. We demonstrate that αT-catenin is primarily a monomer in solution and that αT-catenin monomer binds F-actin in cosedimentation assays as strongly as αE-catenin homodimer. The β-catenin·αT-catenin heterocomplex also binds F-actin with high affinity unlike the β-catenin·αE-catenin complex, indicating that αT-catenin can directly link the cadherin·catenin complex to the actin cytoskeleton. Finally, we show that a mutation in αT-catenin linked to arrhythmogenic right ventricular cardiomyopathy, V94D, promotes homodimerization, blocks β-catenin binding, and in cardiomyocytes disrupts localization at cell-cell contacts. Together, our data demonstrate that αT-catenin is a constitutively active actin-binding protein that can physically couple the cadherin·catenin complex to F-actin in the absence of tension. We speculate that these properties are optimized to meet the demands of cardiomyocyte adhesion. PMID:27231342

  9. Thin Filament Mutations: Developing An Integrative Approach to a Complex Disorder

    PubMed Central

    Tardiff, Jil C.

    2010-01-01

    Sixteen years ago, mutations in cardiac TnT and α-tropomyosin were linked to Familial Hypertrophic Cardiomypathy, thus transforming the disorder from a disease of the β-myosin heavy chain to a disease of the cardiac sarcomere. From the outset, studies suggested that mutations in the regulatory thin filament caused a complex, heterogeneous pattern of ventricular remodeling with wide variations in clinical expression. To date, the clinical heterogeneity is well matched by an extensive array of nearly 100 independent mutations in all components of the cardiac thin filament. Significant advances in our understanding of the biophysics of myofilament activation coupled to the emerging evidence that thin filament linked cardiomyopathies are progressive suggests that a renewed focus on the most proximal events in both the molecular and clinical pathogenesis of the disease will be necessary to achieve the central goal of using genotype information to manage affected patients. In this review we will examine the existing biophysical and clinical evidence in support of a more proximal definition of thin filament cardiomyopathies. In addition, new high resolution, integrated approaches will be presented to help define the way forward as the field works towards developing a more robust link between genotype and phenotype in this complex disorder. PMID:21415410

  10. Genetics Home Reference: actin-accumulation myopathy

    MedlinePlus

    ... fibers and are important for muscle contraction. Attachment (binding) and release of the overlapping thick and thin filaments allows them to move relative to each other so that the muscles can contract. ACTA1 gene mutations that cause actin-accumulation myopathy ...

  11. Structural polymorphism of the actin-espin system: a prototypical system of filaments and linkers in stereocilia.

    PubMed

    Purdy, Kirstin R; Bartles, James R; Wong, Gerard C L

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  12. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin R.; Bartles, James R.; Wong, Gerard C. L.

    2007-02-01

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system’s phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  13. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    SciTech Connect

    Purdy, Kirstin R.; Wong, Gerard C. L.; Bartles, James R.

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  14. Invertebrate muscles: thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle.

    PubMed

    Hooper, Scott L; Hobbs, Kevin H; Thuma, Jeffrey B

    2008-10-01

    This is the second in a series of canonical reviews on invertebrate muscle. We cover here thin and thick filament structure, the molecular basis of force generation and its regulation, and two special properties of some invertebrate muscle, catch and asynchronous muscle. Invertebrate thin filaments resemble vertebrate thin filaments, although helix structure and tropomyosin arrangement show small differences. Invertebrate thick filaments, alternatively, are very different from vertebrate striated thick filaments and show great variation within invertebrates. Part of this diversity stems from variation in paramyosin content, which is greatly increased in very large diameter invertebrate thick filaments. Other of it arises from relatively small changes in filament backbone structure, which results in filaments with grossly similar myosin head placements (rotating crowns of heads every 14.5 nm) but large changes in detail (distances between heads in azimuthal registration varying from three to thousands of crowns). The lever arm basis of force generation is common to both vertebrates and invertebrates, and in some invertebrates this process is understood on the near atomic level. Invertebrate actomyosin is both thin (tropomyosin:troponin) and thick (primarily via direct Ca(++) binding to myosin) filament regulated, and most invertebrate muscles are dually regulated. These mechanisms are well understood on the molecular level, but the behavioral utility of dual regulation is less so. The phosphorylation state of the thick filament associated giant protein, twitchin, has been recently shown to be the molecular basis of catch. The molecular basis of the stretch activation underlying asynchronous muscle activity, however, remains unresolved.

  15. Invertebrate muscles: thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

    PubMed Central

    Hooper, Scott L.; Hobbs, Kevin H.; Thuma, Jeffrey B.

    2008-01-01

    This is the second in a series of canonical reviews on invertebrate muscle. We cover here thin and thick filament structure, the molecular basis of force generation and its regulation, and two special properties of some invertebrate muscle, catch and asynchronous muscle. Invertebrate thin filaments resemble vertebrate thin filaments, although helix structure and tropomyosin arrangement show small differences. Invertebrate thick filaments, alternatively, are very different from vertebrate striated thick filaments and show great variation within invertebrates. Part of this diversity stems from variation in paramyosin content, which is greatly increased in very large diameter invertebrate thick filaments. Other of it arises from relatively small changes in filament backbone structure, which results in filaments with grossly similar myosin head placements (rotating crowns of heads every 14.5 nm) but large changes in detail (distances between heads in azimuthal registration varying from three to thousands of crowns). The lever arm basis of force generation is common to both vetebrates and invertebrates, and in some invertebrates this process is understood on the near atomic level. Invertebrate actomyosin is both thin (tropomyosin:troponin) and thick (primarily via direct Ca++ binding to myosin) filament regulated, and most invertebrate muscles are dually regulated. These mechanisms are well understood on the molecular level, but the behavioral utility of dual regulation is less so. The phosphorylation state of the thick filament associated giant protein, twitchin, has been recently shown to be the molecular basis of catch. The molecular basis of the stretch activation underlying asynchronous muscle activity, however, remains unresolved. PMID:18616971

  16. 12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells

    SciTech Connect

    Shiba, Yoshiki; Sasaki, Yasuto; Kanno, Yoshinobu )

    1988-10-01

    The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodomine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.

  17. The Human Arp2/3 Complex Is Composed of Evolutionarily Conserved Subunits and Is Localized to Cellular Regions of Dynamic Actin Filament Assembly

    PubMed Central

    Welch, Matthew D.; DePace, Angela H.; Verma, Suzie; Iwamatsu, Akihiro; Mitchison, Timothy J.

    1997-01-01

    The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Arp complex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat–containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion. PMID:9230079

  18. The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.

    PubMed

    Welch, M D; DePace, A H; Verma, S; Iwamatsu, A; Mitchison, T J

    1997-07-28

    The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

  19. Heat transfer mechanism with thin filaments including ceramic high temperature heat exchanger

    DOEpatents

    Im, Kwan H.; Ahluwalia, Rajesh K.

    1994-01-01

    A radiative heat transfer mechanism in a furnace having burners through which pulverized coal and air are burned producing combustion gases and contaminants. A plurality of elongated conduits are positioned inside the furnace proximate to the burners generally parallel to the flow of combustion gases in the furnace. A plurality of thin filaments are inside each of the elongated hollow conduits, the filaments having diameters in the range of from about 1 micrometer to about 1,000 micrometers and having an infrared radiation cross-section sufficient to cause the filaments to heat upon exposure to infrared radiation. Blower mechanism is associated with the elongated conduits for limiting the amount of soot and ash which deposit on the conduits to preserve the radiative and convective transfer of heat energy from the combustion gases to the conduits.

  20. Heat transfer mechanism with thin filaments including ceramic high temperature heat exchanger

    DOEpatents

    Im, K.H.; Ahluwalia, R.K.

    1994-10-18

    A radiative heat transfer mechanism in a furnace is described having burners through which pulverized coal and air are burned producing combustion gases and contaminants. A plurality of elongated conduits are positioned inside the furnace proximate to the burners generally parallel to the flow of combustion gases in the furnace. A plurality of thin filaments are inside each of the elongated hollow conduits, the filaments having diameters in the range of from about 1 micrometer to about 1,000 micrometers and having an infrared radiation cross-section sufficient to cause the filaments to heat upon exposure to infrared radiation. Blower mechanism is associated with the elongated conduits for limiting the amount of soot and ash which deposit on the conduits to preserve the radiative and convective transfer of heat energy from the combustion gases to the conduits. 7 figs.

  1. Actin filament-associated protein 1 is required for cSrc activity and secretory activation in the lactating mammary gland

    PubMed Central

    Cunnick, Jess M; Kim, Stephanie; Hadsell, James; Collins, Stephen; Cerra, Carmine; Reiser, Patti; Flynn, Daniel C; Cho, Youngjin

    2014-01-01

    Actin filament-associated protein 1 (AFAP1) is an adaptor protein of cSrc that binds to filamentous actin and regulates the activity of this tyrosine kinase to affect changes to the organization of the actin cytoskeleton. In breast and prostate cancer cells, AFAP1 has been shown to regulate cellular responses requiring actin cytoskeletal changes such as adhesion, invadopodia formation and invasion. However, a normal physiological role for AFAP1 has remained elusive. In this study, we generated an AFAP1 knockout mouse model that establishes a novel physiological role for AFAP1 in lactation. Specifically, these animals displayed a defect in lactation that resulted in an inability to efficiently nurse. Histologically, the mammary glands of the lactating knockout mice were distinguished by the accumulation of large cytoplasmic lipid droplets in the alveolar epithelial cells. There was a reduction in lipid synthesis and the expression of lipogenic genes without a corresponding reduction in the production of beta-casein, a milk protein. Furthermore, these defects were associated with histological and biochemical signs of precocious involution. This study also demonstrated that AFAP1 responds to prolactin, a lactogenic hormone, by forming a complex with cSrc and becoming tyrosine phosphorylated. Together, these observations pointed to a defect in secretory activation. Certain characteristics of this phenotype mirrored the defect in secretory activation in the cSrc knockout mouse, but most importantly, the activity of cSrc in the mammary gland was reduced during early lactation in the AFAP1 null mouse and the localization of active cSrc at the apical surface of luminal epithelial cells during lactation was selectively lost in the absence of AFAP1. These data define, for the first time, the requirement of AFAP1 for the spatial and temporal regulation of cSrc activity in the normal breast, specifically for milk production. PMID:25043309

  2. An explicitly solvated full atomistic model of the cardiac thin filament and application on the calcium binding affinity effects from familial hypertrophic cardiomyopathy linked mutations

    NASA Astrophysics Data System (ADS)

    Williams, Michael; Schwartz, Steven

    2015-03-01

    The previous version of our cardiac thin filament (CTF) model consisted of the troponin complex (cTn), two coiled-coil dimers of tropomyosin (Tm), and 29 actin units. We now present the newest revision of the model to include explicit solvation. The model was developed to continue our study of genetic mutations in the CTF proteins which are linked to familial hypertrophic cardiomyopathies. Binding of calcium to the cTnC subunit causes subtle conformational changes to propagate through the cTnC to the cTnI subunit which then detaches from actin. Conformational changes propagate through to the cTnT subunit, which allows Tm to move into the open position along actin, leading to muscle contraction. Calcium disassociation allows for the reverse to occur, which results in muscle relaxation. The inclusion of explicit TIP3 water solvation allows for the model to get better individual local solvent to protein interactions; which are important when observing the N-lobe calcium binding pocket of the cTnC. We are able to compare in silica and in vitro experimental results to better understand the physiological effects from mutants, such as the R92L/W and F110V/I of the cTnT, on the calcium binding affinity compared to the wild type.

  3. Impact of tropomyosin isoform composition on fast skeletal muscle thin filament regulation and force development.

    PubMed

    Scellini, B; Piroddi, N; Flint, G V; Regnier, M; Poggesi, C; Tesi, C

    2015-02-01

    Tropomyosin (Tm) plays a central role in the regulation of muscle contraction and is present in three main isoforms in skeletal and cardiac muscles. In the present work we studied the functional role of α- and βTm on force development by modifying the isoform composition of rabbit psoas skeletal muscle myofibrils and of regulated thin filaments for in vitro motility measurements. Skeletal myofibril regulatory proteins were extracted (78%) and replaced (98%) with Tm isoforms as homogenous ααTm or ββTm dimers and the functional effects were measured. Maximal Ca(2+) activated force was the same in ααTm versus ββTm myofibrils, but ββTm myofibrils showed a marked slowing of relaxation and an impairment of regulation under resting conditions compared to ααTm and controls. ββTm myofibrils also showed a significantly shorter slack sarcomere length and a marked increase in resting tension. Both these mechanical features were almost completely abolished by 10 mM 2,3-butanedione 2-monoxime, suggesting the presence of a significant degree of Ca(2+)-independent cross-bridge formation in ββTm myofibrils. Finally, in motility assay experiments in the absence of Ca(2+) (pCa 9.0), complete regulation of thin filaments required greater ββTm versus ααTm concentrations, while at full activation (pCa 5.0) no effect was observed on maximal thin filament motility speed. We infer from these observations that high contents of ββTm in skeletal muscle result in partial Ca(2+)-independent activation of thin filaments at rest, and longer-lasting and less complete tension relaxation following Ca(2+) removal.

  4. KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy

    PubMed Central

    Garg, Ankit; O’Rourke, Jason; Long, Chengzu; Doering, Jonathan; Ravenscroft, Gianina; Bezprozvannaya, Svetlana; Nelson, Benjamin R.; Beetz, Nadine; Li, Lin; Chen, She; Laing, Nigel G.; Grange, Robert W.; Bassel-Duby, Rhonda; Olson, Eric N.

    2014-01-01

    Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40–/– mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients. PMID:24960163

  5. Bulk cytoplasmic actin and its functions in meiosis and mitosis.

    PubMed

    Field, Christine M; Lénárt, Péter

    2011-10-11

    Discussions of actin cell biology generally focus on the cortex, a thin, actin-rich layer of cytoplasm under the plasma membrane. Here we review the much less studied biology of actin filaments deeper in the cytoplasm and their recently revealed functions in mitosis and meiosis that are most prominent in large oocyte, egg and early embryo cells. The cellular functions of cytoplasmic actin range from the assembly and positioning of meiotic spindles to the prevention of cytoplasmic streaming. We discuss the possible use of evolutionarily conserved mechanisms to nucleate and organize actin filaments to achieve these diverse cellular functions, the cell-cycle regulation of these functions, and the many unanswered questions about this largely unexplored mechanism of cytoplasmic organization.

  6. Using the peptide BP100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells.

    PubMed

    Eggenberger, Kai; Mink, Christian; Wadhwani, Parvesh; Ulrich, Anne S; Nick, Peter

    2011-01-03

    The delivery of externally applied macromolecules or nanoparticles into living cells still represents a critically limiting step before the full capabilities of chemical engineering can be explored. Molecular transporters such as cell-penetrating peptides, peptoids, and other mimetics can be used to carry cargo across the cellular membrane, but it is still difficult to find suitable sequences that operate efficiently for any particular type of cell. Here we report that BP100 (KKLFKKILKYL-amide), originally designed as an antimicrobial peptide against plant pathogens, can be employed as a fast and efficient cell-penetrating agent to transport fluorescent test cargoes into the cytosol of walled plant cells. The uptake of BP100 proceeds slightly more slowly than the endocytosis of fluorescent dextranes, but BP100 accumulates more efficiently and to much higher levels (by an order of magnitude). The entry of BP100 can be efficiently blocked by latrunculin B; this suggests that actin filaments are essential to the uptake mechanism. To test whether this novel transporter can also be used to deliver functional cargoes, we designed a fusion construct of BP100 with the actin-binding Lifeact peptide (MGVADLIKKFESISKEE). We demonstrated that the short BP100 could transport the attached 17-residue sequence quickly and efficiently into tobacco cells. The Lifeact construct retained its functionality as it successfully labeled the actin bundles that tether the nucleus in the cell center.

  7. Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements.

    PubMed

    Buxa, Stefanie V; Degola, Francesca; Polizzotto, Rachele; De Marco, Federica; Loschi, Alberto; Kogel, Karl-Heinz; di Toppi, Luigi Sanità; van Bel, Aart J E; Musetti, Rita

    2015-01-01

    Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by 'Candidatus Phytoplasma solani,' the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes.

  8. CARMIL is a potent capping protein antagonist: identification of a conserved CARMIL domain that inhibits the activity of capping protein and uncaps capped actin filaments.

    PubMed

    Uruno, Takehito; Remmert, Kirsten; Hammer, John A

    2006-04-14

    Acanthamoeba CARMIL was previously shown to co-purify with capping protein (CP) and to bind pure CP. Here we show that this interaction inhibits the barbed end-capping activity of CP. Even more strikingly, this interaction drives the uncapping of actin filaments previously capped with CP. These activities are CP-specific; CARMIL does not inhibit the capping activities of either gelsolin or CapG and does not uncap gelsolin-capped filaments. Although full-length (FL) CARMIL (residues 1-1121) possesses both anti-CP activities, C-terminal fragments like glutathione S-transferase (GST)-P (940-1121) that contain the CARMIL CP binding site are at least 10 times more active. We localized the full activities of GST-P to its C-terminal 51 residues (1071-1121). This sequence contains a stretch of 25 residues that is highly conserved in CARMIL proteins from protozoa, flies, worms, and vertebrates (CARMIL Homology domain 3; CAH3). Point mutations showed that the majority of the most highly conserved residues within CAH3 are critical for the anti-CP activity of GST-AP (862-1121). Finally, we found that GST-AP binds CP approximately 20-fold more tightly than does FL-CARMIL. This observation together with the elevated activities of C-terminal fragments relative to FL-CARMIL suggests that FL-CARMIL might exist primarily in an autoinhibited state. Consistent with this idea, proteolytic cleavage of FL-CARMIL with thrombin generated an approximately 14-kDa C-terminal fragment that expresses full anti-CP activities. We propose that, after some type of physiological activation event, FL-CARMIL could function in vivo as a potent CP antagonist. Given the pivotal role that CP plays in determining the global actin phenotype of cells, our results suggest that CARMIL may play an important role in the physiological regulation of actin assembly.

  9. Slowed Dynamics of Thin Filament Regulatory Units Reduces Ca2+-Sensitivity of Cardiac Biomechanical Function

    PubMed Central

    Loong, Campion K. P.; Takeda, Aya K.; Badr, Myriam A.; Rogers, Jordan S.; Chase, P. Bryant

    2013-01-01

    Actomyosin kinetics in both skinned skeletal muscle fibers at maximum Ca2+-activation and unregulated in vitro motility assays are modulated by solvent microviscosity in a manner consistent with a diffusion limited process. Viscosity might also influence cardiac thin filament Ca2+-regulatory protein dynamics. In vitro motility assays were conducted using thin filaments reconstituted with recombinant human cardiac troponin and tropomyosin; solvent microviscosity was varied by addition of sucrose or glucose. At saturating Ca2+, filament sliding speed (s) was inversely proportional to viscosity. Ca2+-sensitivity (pCa50) of s decreased markedly with elevated viscosity (η/η0 ≥ ~1.3). For comparison with unloaded motility assays, steady-state isometric force (F) and kinetics of isometric tension redevelopment (kTR) were measured in single, permeabilized porcine cardiomyocytes when viscosity surrounding the myofilaments was altered. Maximum Ca2+-activated F changed little for sucrose ≤ 0.3 M (η/η0 ~1.4) or glucose ≤ 0.875 M (η/η0 ~1.66), but decreased at higher concentrations. Sucrose (0.3 M) or glucose (0.875 M) decreased pCa50 for F. kTR at saturating Ca2+ decreased steeply and monotonically with increased viscosity but there was little effect on kTR at sub-maximum Ca2+. Modeling indicates that increased solutes affect dynamics of cardiac muscle Ca2+-regulatory proteins to a much greater extent than actomyosin cross-bridge cycling. PMID:23833690

  10. Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

    PubMed Central

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P.; Kumar, Mohit; Greaser, Marion L.; Irving, Thomas C.; de Tombe, Pieter P.

    2016-01-01

    The Frank–Starling mechanism of the heart is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank–Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA. PMID:26858417

  11. Titin strain contributes to the Frank-Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins.

    PubMed

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P; Kumar, Mohit; Greaser, Marion L; Irving, Thomas C; de Tombe, Pieter P

    2016-02-23

    The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca(2+) sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.

  12. Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

    SciTech Connect

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P.; Kumar, Mohit; Greaser, Marion L.; Irving, Thomas C.; de Tombe, Pieter P.

    2016-02-08

    The Frank–Starling mechanism of the heart is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank–Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.

  13. Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

    DOE PAGES

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P.; ...

    2016-02-08

    The Frank–Starling mechanism of the heart is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT musclesmore » displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank–Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.« less

  14. Titin strain contributes to the Frank-Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

    SciTech Connect

    Ait-Mou, Younss; Hsu, Karen; Farman, Gerrie P.; Kumar, Mohit; Greaser, Marion L.; Irving, Thomas C.; de Tombe, Pieter P.

    2016-04-29

    We determined that the Frank–Starling mechanism of the heart is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Our results reveal a correlation between titin strain and the Frank–Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.

  15. Initial binding of Shiga toxin-producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA-containing surface appendages.

    PubMed

    Ebel, F; Podzadel, T; Rohde, M; Kresse, A U; Krämer, S; Deibel, C; Guzmán, C A; Chakraborty, T

    1998-10-01

    Shiga toxin-producing Escherichia coli (STEC) induce so-called attaching and effacing lesions that enable the tight adherence of these pathogens to the gut epithelium. All of the genes necessary for this process are present in the locus of enterocyte effacement, which encodes a type III secretion system, the secreted Esp proteins and the surface protein intimin. In this study we sequenced the espA gene of STEC, generated and characterized a corresponding deletion mutant and raised EspA-specific monoclonal antibodies to analyse the functional role of this protein during infection. EspA was detected in often filament-like structures decorating all bacteria that had attached to HeLa cells. These appendages were especially prominent on bacteria that had not yet induced the formation of actin pedestals, indicating that they mediate the initial contact of STEC to their target cells. Consistently, a deletion of the espA gene completely abolished the capacity of such STEC mutants to bind to HeLa cells and to induce actin rearrangements. Surface appendages similar to those described in this study are also formed by Pseudomonas syringae and may represent a structural element common to many bacterial pathogens that deliver proteins into their target cells via a type III secretion system.

  16. Structural Evidence for Actin-like Filaments in Toxoplasma gondii Using High-Resolution Low-Voltage Field Emission Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Schatten, Heide; Sibley, L. David; Ris, Hans

    2003-08-01

    The protozoan parasite Toxoplasma gondii is representative of a large group of parasites within the phylum Apicomplexa, which share a highly unusual motility system that is crucial for locomotion and active host cell invasion. Despite the importance of motility in the pathology of these unicellular organisms, the motor mechanisms for locomotion remain uncertain, largely because only limited data exist about composition and organization of the cytoskeleton. By using cytoskeleton stabilizing protocols on membrane-extracted parasites and novel imaging with high-resolution low-voltage field emission scanning electron microscopy (LVFESEM), we were able to visualize for the first time a network of actin-sized filaments just below the cell membrane. A complex cytoskeletal network remained after removing the actin-sized fibers with cytochalasin D, revealing longitudinally arranged, subpellicular microtubules and intermediate-sized fibers of 10 nm, which, in stereo images, are seen both above and below the microtubules. These approaches open new possibilities to characterize more fully the largely unexplored and unconventional cytoskeletal motility complex in apicomplexan parasites.

  17. Titin isoform size is not correlated with thin filament length in rat skeletal muscle

    PubMed Central

    Greaser, Marion L.; Pleitner, Jonathan M.

    2014-01-01

    The mechanisms controlling thin filament length (TFL) in muscle remain controversial. It was recently reported that TFL was related to titin size, and that the latter might be involved in TFL determination. Titin plays several crucial roles in the sarcomere, but its function as it pertains to the thin filament has not been explored. We tested this relationship using several muscles from wild type rats and from a mutant rat model (Greaser et al., 2008) which results in increased titin size. Myofibrils were isolated from skeletal muscles [extensor digitorum longus (EDL), external oblique (EO), gastrocnemius (GAS), longissimus dorsi (LD), psoas major (PM), and tibialis anterior(TA)] using both adult wild type (WT) and homozygous mutant (HM) rats (n = 6 each). Phalloidin and antibodies against tropomodulin-4 (Tmod-4) and nebulin's N-terminus were used to determine TFL. The WT rats studied express skeletal muscle titin sizes ranging from 3.2 to 3.7 MDa, while the HM rats express a giant titin isoform sized at 3.8 MDa. No differences in phalloidin based TFL, nebulin distance, or Tmod distance were observed across genotypes. However, the HM rats demonstrated a significantly increased (p < 0.01) rest sarcomere length relative to the WT phenotype. It appears that the increased titin size, predominantly observed in HM rats' middle Ig domain, allows for increased extensibility. The data indicates that, although titin performs many sarcomeric functions, its correlation with TFL and structure could not be demonstrated in the rat. PMID:24550844

  18. Ising model of cardiac thin filament activation with nearest-neighbor cooperative interactions

    NASA Technical Reports Server (NTRS)

    Rice, John Jeremy; Stolovitzky, Gustavo; Tu, Yuhai; de Tombe, Pieter P.; Bers, D. M. (Principal Investigator)

    2003-01-01

    We have developed a model of cardiac thin filament activation using an Ising model approach from equilibrium statistical physics. This model explicitly represents nearest-neighbor interactions between 26 troponin/tropomyosin units along a one-dimensional array that represents the cardiac thin filament. With transition rates chosen to match experimental data, the results show that the resulting force-pCa (F-pCa) relations are similar to Hill functions with asymmetries, as seen in experimental data. Specifically, Hill plots showing (log(F/(1-F)) vs. log [Ca]) reveal a steeper slope below the half activation point (Ca(50)) compared with above. Parameter variation studies show interplay of parameters that affect the apparent cooperativity and asymmetry in the F-pCa relations. The model also predicts that Ca binding is uncooperative for low [Ca], becomes steeper near Ca(50), and becomes uncooperative again at higher [Ca]. The steepness near Ca(50) mirrors the steep F-pCa as a result of thermodynamic considerations. The model also predicts that the correlation between troponin/tropomyosin units along the one-dimensional array quickly decays at high and low [Ca], but near Ca(50), high correlation occurs across the whole array. This work provides a simple model that can account for the steepness and shape of F-pCa relations that other models fail to reproduce.

  19. A new member of the Rho family, Rnd1, promotes disassembly of actin filament structures and loss of cell adhesion.

    PubMed

    Nobes, C D; Lauritzen, I; Mattei, M G; Paris, S; Hall, A; Chardin, P

    1998-04-06

    Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell-substrate adhesion leading to cell rounding (hence Rnd for "round"). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.

  20. Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

    PubMed Central

    Arden, Susan D.; Tumbarello, David A.; Butt, Tariq; Kendrick-Jones, John; Buss, Folma

    2016-01-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability. PMID:27474411

  1. Filamentous fungal-specific septin AspE is phosphorylated in vivo and interacts with actin, tubulin and other septins in the human pathogen Aspergillus fumigatus

    SciTech Connect

    Juvvadi, Praveen Rao; Belina, Detti; Soderblom, Erik J.; Moseley, M. Arthur; Steinbach, William J.

    2013-02-15

    Highlights: ► In vivo interactions of the novel septin AspE were identified by GFP-Trap® affinity purification. ► Septins AspA, AspB, AspC and AspD interacted with AspE in vivo. ► Actin and tubulin interacted with AspE in vivo. ► AspE is phosphorylated at six serine residues in vivo. -- Abstract: We previously analyzed the differential localization patterns of five septins (AspA–E), including a filamentous fungal-specific septin, AspE, in the human pathogen Aspergillus fumigatus. Here we utilized the A. fumigatus strain expressing an AspE–EGFP fusion protein and show that this novel septin with a tubular localization pattern in hyphae is phosphorylated in vivo and interacts with the other septins, AspA, AspB, AspC and AspD. The other major proteins interacting with AspE included the cytoskeletal proteins, actin and tubulin, which may be involved in the organization and transport of the septins. This is the first report analyzing the phosphorylation of AspE and localizing the sites of phosphorylation, and opens opportunities for further analysis on the role of post-translational modifications in the assembly and organization of A. fumigatus septins. This study also describes the previously unknown interaction of AspE with the actin-microtubule network. Furthermore, the novel GFP-Trap® affinity purification method used here complements widely-used GFP localization studies in fungal systems.

  2. Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation

    NASA Astrophysics Data System (ADS)

    Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George

    2012-03-01

    With traditional 2-color Förster Resonance Energy Transfer (FRET) microscopy, valuable quantitative analyses can be conducted. Correlations of donor (D), acceptor (A) and their ratios (D:A) with energy transfer efficiency (E%) or distance (r) allows measurement of changes between control and experimental samples; also, clustered vs. random assembly of cellular components can be differentiated. Essentially, only the above three parameters D, A and D:A vs. E% are the basis for these deductions. 3-color FRET uses the same basic parameters, but exponentially expands the opportunities to quantify interrelationships among 3 cellular components. We investigated a number of questions based on the results of a triple combination (F1-F2-F3) of TFPNWASP/ Venus-IQGAP1/mCherry-Actin - all involved in the nucleation of actin - to apply the extensive analysis assay possible with 3-color FRET. How do changing N-WASP or IQGAP1 fluorescence levels affect actin fluorescence? What is the effect on E% of NWASP-actin by IQGAP1 or E% of IQGAP1-actin by N-WASP? These and other questions are explored in the context of all proteins of interest being in FRET distance vs. any two in the absence of the third. 4 cases are compared based on bleed-through corrected FRET: (1) all 3 interact, (2) only F1- F3 and F2-F3 [not F1-F2], (3) only F1-F2 and F2-F3 interact [not F1-F3], (4) only F1-F2 and F1-F3 interact [not F2-F3]. Other than describing the methodology in detail, several biologically relevant results are presented showing how E% (i.e. distance), fluorescence levels and ratios are affected in each of the cases. These correlations can only be observed in a 3-fluorophore combination. 3-color FRET will greatly expand the investigative range of quantitative analysis for the life-science researcher.

  3. Why is Actin Patchy?

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2009-03-01

    The intracellular protein actin, by reversibly polymerizing into filaments, generates forces for motion and shape changes of many types of biological cells. Fluorescence imaging studies show that actin often occurs in the form of localized patches of size roughly one micrometer at the cell membrane. Patch formation is most prevalent when the free-actin concentration is low. I investigate possible mechanisms for the formation of actin patches by numerically simulating the ``dendritic nucleation'' model of actin network growth. The simulations include filament growth, capping, branching, severing, and debranching. The attachment of membrane-bound activators to actin filaments, and subsequent membrane diffusion of unattached activators, are also included. It is found that as the actin concentration increases from zero, the actin occurs in patches at lower actin concentrations, and the size of the patches increases with increasing actin concentration. At a critical value of the actin concentration, the system undergoes a transition to complete coverage. The results are interpreted within the framework of reaction-diffusion equations in two dimensions.

  4. An atomic model of the tropomyosin cable on F-actin.

    PubMed

    Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan; Lehman, William

    2014-08-05

    Tropomyosin regulates a wide variety of actin filament functions and is best known for the role that it plays together with troponin in controlling muscle activity. For effective performance on actin filaments, adjacent 42-nm-long tropomyosin molecules are joined together by a 9- to 10-residue head-to-tail overlapping domain to form a continuous cable that wraps around the F-actin helix. Yet, despite the apparent simplicity of tropomyosin's coiled-coil structure and its well-known periodic association with successive actin subunits along F-actin, the structure of the tropomyosin cable on actin is uncertain. This is because the conformation of the overlap region that joins neighboring molecules is poorly understood, thus leaving a significant gap in our understanding of thin-filament structure and regulation. However, recent molecular-dynamics simulations of overlap segments defined their overall shape and provided unique and sufficient cues to model the whole actin-tropomyosin filament assembly in atomic detail. In this study, we show that these MD structures merge seamlessly onto the ends of tropomyosin coiled-coils. Adjacent tropomyosin molecules can then be joined together to provide a comprehensive model of the tropomyosin cable running continuously on F-actin. The resulting complete model presented here describes for the first time (to our knowledge) an atomic-level structure of αα-striated muscle tropomyosin bound to an actin filament that includes the critical overlap domain. Thus, the model provides a structural correlate to evaluate thin-filament mechanics, self-assembly mechanisms, and the effect of disease-causing mutations.

  5. Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics.

    PubMed

    Speranza, Luisa; Giuliano, Teresa; Volpicelli, Floriana; De Stefano, M Egle; Lombardi, Loredana; Chambery, Angela; Lacivita, Enza; Leopoldo, Marcello; Bellenchi, Gian C; di Porzio, Umberto; Crispino, Marianna; Perrone-Capano, Carla

    2015-01-01

    Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

  6. Uncorrelated multiple conductive filament nucleation and rupture in ultra-thin high-κ dielectric based resistive random access memory

    NASA Astrophysics Data System (ADS)

    Wu, Xing; Li, Kun; Raghavan, Nagarajan; Bosman, Michel; Wang, Qing-Xiao; Cha, Dongkyu; Zhang, Xi-Xiang; Pey, Kin-Leong

    2011-08-01

    Resistive switching in transition metal oxides could form the basis for next-generation non-volatile memory (NVM). It has been reported that the current in the high-conductivity state of several technologically relevant oxide materials flows through localized filaments, but these filaments have been characterized only individually, limiting our understanding of the possibility of multiple conductive filaments nucleation and rupture and the correlation kinetics of their evolution. In this study, direct visualization of uncorrelated multiple conductive filaments in ultra-thin HfO2-based high-κ dielectric resistive random access memory (RRAM) device has been achieved by high-resolution transmission electron microscopy (HRTEM), along with electron energy loss spectroscopy (EELS), for nanoscale chemical analysis. The locations of these multiple filaments are found to be spatially uncorrelated. The evolution of these microstructural changes and chemical properties of these filaments will provide a fundamental understanding of the switching mechanism for RRAM in thin oxide films and pave way for the investigation into improving the stability and scalability of switching memory devices.

  7. Role of Active Contraction and Tropomodulins in Regulating Actin Filament Length and Sarcomere Structure in Developing Zebrafish Skeletal Muscle

    PubMed Central

    Mazelet, Lise; Parker, Matthew O.; Li, Mei; Arner, Anders; Ashworth, Rachel

    2016-01-01

    Whilst it is recognized that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1ts25) which lacks functional voltage-gated calcium channels (dihydropyridine receptors) in the muscle and pharmacological immobilization of embryos with a reversible anesthetic (Tricaine), allowed the study of paralysis (in mutants and anesthetized fish) and recovery of movement (reversal of anesthetic treatment). The effect of paralysis in early embryos (aged between 17 and 24 hours post-fertilization, hpf) on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localization of the actin capping proteins Tropomodulin 1 & 4 (Tmod) in fish aged from 17 hpf until 42 hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post-fertilization (dpf). Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf) resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralyzed fish by 42 hpf. In conclusion, myofibril organization is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localization of Tmod1 to its sarcomeric position

  8. Extracting the ridge set as a graph for actin filament length estimation from confocal laser scanning microscopic images

    NASA Astrophysics Data System (ADS)

    Birkholz, Harald

    2012-04-01

    The progress in image acquisition techniques provides life sciences with an abundance of data. Image analysis facilitates the assessment. The actin cytoskeleton plays a crucial role in understanding the behavior of osteoblastic cells on biomaterials. In the flat basal part of the cells, it can be visualized by confocal laser scanning microscopy. In the microscopic images, the stained cytoskeleton appears as a dense network of bright ridges which is so far only qualitatively assessed. For its quantification, there is a need for ridge detection techniques that provide a geometrical description of this graph feature. The state of the art methods do not cope with the systematical degradation by noise, unspecific luminance, and uneven dye uptake. This work presents the key part of a ridge-tracking technique, which makes more efficient use of context information, and evaluate it by its length measurement accuracy. Two random models illustrate the performance against ground truth. Representative microscopic images confirm the applicability.

  9. Thick-to-Thin Filament Surface Distance Modulates Cross-Bridge Kinetics in Drosophila Flight Muscle

    SciTech Connect

    Tanner, Bertrand C.W.; Farman, Gerrie P.; Irving, Thomas C.; Maughan, David W.; Palmer, Bradley M.; Miller, Mark S.

    2012-09-19

    The demembranated (skinned) muscle fiber preparation is widely used to investigate muscle contraction because the intracellular ionic conditions can be precisely controlled. However, plasma membrane removal results in a loss of osmotic regulation, causing abnormal hydration of the myofilament lattice and its proteins. We investigated the structural and functional consequences of varied myofilament lattice spacing and protein hydration on cross-bridge rates of force development and detachment in Drosophila melanogaster indirect flight muscle, using x-ray diffraction to compare the lattice spacing of dissected, osmotically compressed skinned fibers to native muscle fibers in living flies. Osmolytes of different sizes and exclusion properties (Dextran T-500 and T-10) were used to differentially alter lattice spacing and protein hydration. At in vivo lattice spacing, cross-bridge attachment time (t{sub on}) increased with higher osmotic pressures, consistent with a reduced cross-bridge detachment rate as myofilament protein hydration decreased. In contrast, in the swollen lattice, t{sub on} decreased with higher osmotic pressures. These divergent responses were reconciled using a structural model that predicts t{sub on} varies inversely with thick-to-thin filament surface distance, suggesting that cross-bridge rates of force development and detachment are modulated more by myofilament lattice geometry than protein hydration. Generalizing these findings, our results suggest that cross-bridge cycling rates slow as thick-to-thin filament surface distance decreases with sarcomere lengthening, and likewise, cross-bridge cycling rates increase during sarcomere shortening. Together, these structural changes may provide a mechanism for altering cross-bridge performance throughout a contraction-relaxation cycle.

  10. Filamentous structures in skeletal muscle: anchors for the subsarcolemmal space.

    PubMed

    Khairani, Astrid Feinisa; Tajika, Yuki; Takahashi, Maiko; Ueno, Hitoshi; Murakami, Tohru; Soenggono, Arifin; Yorifuji, Hiroshi

    2015-03-01

    In skeletal muscle fibers, intermediate filaments and actin filaments provide structural support to the myofibrils and the sarcolemma. For many years, it was poorly understood from ultrastructural observations that how these filamentous structures were kept anchored. The present study was conducted to determine the architecture of filamentous anchoring structures in the subsarcolemmal space and the intermyofibrils. The diaphragms (Dp) of adult wild type and mdx mice (mdx is a model for Duchenne muscular dystrophy) were subjected to tension applied perpendicular to the long axis of the muscle fibers, with or without treatment with 1% Triton X-100 or 0.03% saponin. These experiments were conducted to confirm the presence and integrity of the filamentous anchoring structures. Transmission electron microscopy revealed that these structures provide firm transverse connections between the sarcolemma and peripheral myofibrils. Most of the filamentous structures appeared to be inserted into subsarcolemmal densities, forming anchoring connections between the sarcolemma and peripheral myofibrils. In some cases, actin filaments were found to run longitudinally in the subsarcolemmal space to connect to the sarcolemma or in some cases to connect to the intermyofibrils as elongated thin filaments. These filamentous anchoring structures were less common in the mdx Dp. Our data suggest that the transverse and longitudinal filamentous structures form an anchoring system in the subsarcolemmal space and the intermyofibrils.

  11. Evidence for {gamma}-actin as a Z disc component in skeletal myofibers

    SciTech Connect

    Papponen, Hinni; Kaisto, Tuula; Leinonen, Sanna; Kaakinen, Mika; Metsikkoe, Kalervo

    2009-01-15

    We investigated the targeting of the {gamma}-actin isoform in skeletal myofibers. For this purpose we used expression vectors to produce green fluorescent protein (GFP-) as well as myc-tagged {gamma}-actin in rat flexor digitorum brevis myofibers. We found that the {gamma}-actin fusion proteins accumulated into Z discs but not beneath the sarcolemma. Instead, the GFP-tagged skeletal muscle-specific {alpha}-actin isoform was preferentially incorporated into the pointed ends of thin contractile filaments. The localization pattern of the {gamma}-actin fusion proteins was completely different from that of the dystrophin glycoprotein complex on the sarcolemma. The results emphasize the role of {gamma}-actin as a Z disc component but fail to reveal an actin-based sub-sarcolemmal cytoskeleton in skeletal muscle cells.

  12. Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

    SciTech Connect

    Wu, Shenping; Liu, Jun; Reedy, Mary C.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A.

    2010-10-22

    Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the 'target zone', situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77{sup o}/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127{sup o} range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very

  13. Thin filament activation probed by fluorescence of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle.

    PubMed

    Brenner, B; Kraft, T; Yu, L C; Chalovich, J M

    1999-11-01

    A method is described for the exchange of native troponin of single rabbit psoas muscle fibers for externally applied troponin complexes without detectable impairment of functional properties of the skinned fibers. This approach is used to exchange native troponin for rabbit skeletal troponin with a fluorescent label (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole, IANBD) on Cys(133) of the troponin I subunit. IANBD-labeled troponin I has previously been used in solution studies as an indicator for the state of activation of reconstituted actin filaments (. Proc. Natl. Acad. Sci. USA. 77:7209-7213). In the skinned fibers, the fluorescence of this probe is unaffected when cross-bridges in their weak binding states attach to actin filaments but decreases either upon the addition of Ca(2+) or when cross-bridges in their strong binding states attach to actin. Maximum reduction is observed when Ca(2+) is raised to saturating concentrations. Additional attachment of cross-bridges in strong binding states gives no further reduction of fluorescence. Attachment of cross-bridges in strong binding states alone (low Ca(2+) concentration) gives only about half of the maximum reduction seen with the addition of calcium. This illustrates that fluorescence of IANBD-labeled troponin I can be used to evaluate thin filament activation, as previously introduced for solution studies. In addition, at nonsaturating Ca(2+) concentrations IANBD fluorescence can be used for straightforward classification of states of the myosin head as weak binding (nonactivating) and strong binding (activating), irrespective of ionic strength or other experimental conditions. Furthermore, the approach presented here not only can be used as a means of exchanging native skeletal troponin and its subunits for a variety of fluorescently labeled or mutant troponin subunits, but also allows the exchange of native skeletal troponin for cardiac troponin.

  14. Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle

    PubMed Central

    Fusi, L.; Brunello, E.; Yan, Z.; Irving, M.

    2016-01-01

    Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway. PMID:27796302

  15. In Situ Time-Resolved FRET Reveals Effects of Sarcomere Length on Cardiac Thin-Filament Activation

    PubMed Central

    Li, King-Lun; Rieck, Daniel; Solaro, R. John; Dong, Wenji

    2014-01-01

    During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca2+ and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca2+-induced opening of N-cTnC and is presumed to also destabilize cTnI–actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca2+-sensitizing N-cTnC–cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca2+-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca2+ and 1.80 and 2.2-μm sarcomere length. Mg2+-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca2+] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback

  16. In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation.

    PubMed

    Li, King-Lun; Rieck, Daniel; Solaro, R John; Dong, Wenji

    2014-08-05

    During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca(2+) and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca(2+)-induced opening of N-cTnC and is presumed to also destabilize cTnI-actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca(2+)-sensitizing N-cTnC-cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca(2+)-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca(2+) and 1.80 and 2.2-μm sarcomere length. Mg(2+)-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca(2+)] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive

  17. Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments.

    PubMed

    Schevzov, Galina; Kee, Anthony J; Wang, Bin; Sequeira, Vanessa B; Hook, Jeff; Coombes, Jason D; Lucas, Christine A; Stehn, Justine R; Musgrove, Elizabeth A; Cretu, Alexandra; Assoian, Richard; Fath, Thomas; Hanoch, Tamar; Seger, Rony; Pleines, Irina; Kile, Benjamin T; Hardeman, Edna C; Gunning, Peter W

    2015-07-01

    ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor-stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.

  18. Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments

    PubMed Central

    Schevzov, Galina; Kee, Anthony J.; Wang, Bin; Sequeira, Vanessa B.; Hook, Jeff; Coombes, Jason D.; Lucas, Christine A.; Stehn, Justine R.; Musgrove, Elizabeth A.; Cretu, Alexandra; Assoian, Richard; Fath, Thomas; Hanoch, Tamar; Seger, Rony; Pleines, Irina; Kile, Benjamin T.; Hardeman, Edna C.; Gunning, Peter W.

    2015-01-01

    ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor–stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells. PMID:25971798

  19. Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM).

    PubMed

    Wang, Yilin; Kanchanawong, Pakorn

    2016-12-01

    Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

  20. Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection.

    PubMed Central

    Valster, A. H.; Pierson, E. S.; Valenta, R.; Hepler, P. K.; Emons, AMC.

    1997-01-01

    We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. Injection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability to act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive to microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells. PMID:12237348

  1. Amplification of actin polymerization forces

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2016-01-01

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments. PMID:27002174

  2. Amplification of actin polymerization forces.

    PubMed

    Dmitrieff, Serge; Nédélec, François

    2016-03-28

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments.

  3. Axonal actin in action: Imaging actin dynamics in neurons.

    PubMed

    Ladt, Kelsey; Ganguly, Archan; Roy, Subhojit

    2016-01-01

    Actin is a highly conserved, key cytoskeletal protein involved in numerous structural and functional roles. In neurons, actin has been intensively investigated in axon terminals-growth cones-and dendritic spines, but details about actin structure and dynamics in axon shafts have remained obscure for decades. A major barrier in the field has been imaging actin. Actin exists as soluble monomers (G-actin) as well as actin filaments (F-actin), and labeling actin with conventional fluorescent probes like GFP/RFP typically leads to a diffuse haze that makes it difficult to discern kinetic behaviors. In a recent publication, we used F-actin selective probes to visualize actin dynamics in axons, resolving striking actin behaviors that have not been described before. However, using these probes to visualize actin dynamics is challenging as they can cause bundling of actin filaments; thus, experimental parameters need to be strictly optimized. Here we describe some practical methodological details related to using these probes for visualizing F-actin dynamics in axons.

  4. Monophasic Pulsed 200-μA Current Promotes Galvanotaxis With Polarization of Actin Filament and Integrin α2β1 in Human Dermal Fibroblasts

    PubMed Central

    Uemura, Mikiko; Maeshige, Noriaki; Koga, Yuka; Ishikawa-Aoyama, Michiko; Miyoshi, Makoto; Sugimoto, Masaharu; Terashi, Hiroto

    2016-01-01

    Objective: The monophasic pulsed microcurrent is used to promote wound healing, and galvanotaxis regulation has been reported as one of the active mechanisms in the promotion of tissue repair with monophasic pulsed microcurrent. However, the optimum monophasic pulsed microcurrent parameters and intracellular changes caused by the monophasic pulsed microcurrent have not been elucidated in human dermal fibroblasts. The purpose of this study was to investigate the optimum intensity for promoting galvanotaxis and the effects of electrical stimulation on integrin α2β1 and actin filaments in human dermal fibroblasts. Methods: Human dermal fibroblasts were treated with the monophasic pulsed microcurrent of 0, 100, 200, or 300 μA for 8 hours, and cell migration and cell viability were measured 24 hours after starting monophasic pulsed microcurrent stimulation. Polarization of integrin α2β1 and lamellipodia formation were detected by immunofluorescent staining 10 minutes after starting monophasic pulsed microcurrent stimulation. Results: The migration toward the cathode was significantly higher in the cells treated with the 200-μA monophasic pulsed microcurrent than in the controls (P < .01) without any change in cell viability; treatment with 300-μA monophasic pulsed microcurrent did not alter the migration ratio. The electrostimulus of 200 μA also promoted integrin α2β1 polarization and lamellipodia formation at the cathode edge (P < .05). Conclusion: The results show that 200 μA is an effective monophasic pulsed microcurrent intensity to promote migration toward the cathode, and this intensity could regulate polarization of migration-related intracellular factors in human dermal fibroblasts. PMID:26819649

  5. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

    NASA Technical Reports Server (NTRS)

    Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

  6. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2’s function

    PubMed Central

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T.; Novak, Stefanie M.; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M.; Helms, Gregory; Gregorio, Carol C.; Kostyukova, Alla S.

    2016-01-01

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2–knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43–90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124–201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly­merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. PMID:27307584

  7. Cooperative cross-bridge activation of thin filaments contributes to the Frank-Starling mechanism in cardiac muscle.

    PubMed

    Smith, L; Tainter, C; Regnier, M; Martyn, D A

    2009-05-06

    Myosin cross-bridges play an important role in the regulation of thin-filament activation in cardiac muscle. To test the hypothesis that sarcomere length (SL) modulation of thin-filament activation by strong-binding cross-bridges underlies the Frank-Starling mechanism, we inhibited force and strong cross-bridge binding to intermediate levels with sodium vanadate (Vi). Force and stiffness varied proportionately with [Ca(2+)] and [Vi]. Increasing [Vi] (decreased force) reduced the pCa(50) of force-[Ca(2+)] relations at 2.3 and 2.0 microm SL, with little effect on slope (n(H)). When maximum force was inhibited to approximately 40%, the effects of SL on force were diminished at lower [Ca(2+)], whereas at higher [Ca(2+)] (pCa < 5.6) the relative influence of SL on force increased. In contrast, force inhibition to approximately 20% significantly reduced the sensitivity of force-[Ca(2+)] relations to changes in both SL and myofilament lattice spacing. Strong cross-bridge binding cooperatively induced changes in cardiac troponin C structure, as measured by dichroism of 5' iodoacetamido-tetramethylrhodamine-labeled cardiac troponin C. This apparent cooperativity was reduced at shorter SL. These data emphasize that SL and/or myofilament lattice spacing modulation of the cross-bridge component of cardiac thin-filament activation contributes to the Frank-Starling mechanism.

  8. Cooperative Cross-Bridge Activation of Thin Filaments Contributes to the Frank-Starling Mechanism in Cardiac Muscle

    PubMed Central

    Smith, L.; Tainter, C.; Regnier, M.; Martyn, D.A.

    2009-01-01

    Myosin cross-bridges play an important role in the regulation of thin-filament activation in cardiac muscle. To test the hypothesis that sarcomere length (SL) modulation of thin-filament activation by strong-binding cross-bridges underlies the Frank-Starling mechanism, we inhibited force and strong cross-bridge binding to intermediate levels with sodium vanadate (Vi). Force and stiffness varied proportionately with [Ca2+] and [Vi]. Increasing [Vi] (decreased force) reduced the pCa50 of force-[Ca2+] relations at 2.3 and 2.0 μm SL, with little effect on slope (nH). When maximum force was inhibited to ∼40%, the effects of SL on force were diminished at lower [Ca2+], whereas at higher [Ca2+] (pCa < 5.6) the relative influence of SL on force increased. In contrast, force inhibition to ∼20% significantly reduced the sensitivity of force-[Ca2+] relations to changes in both SL and myofilament lattice spacing. Strong cross-bridge binding cooperatively induced changes in cardiac troponin C structure, as measured by dichroism of 5′ iodoacetamido-tetramethylrhodamine-labeled cardiac troponin C. This apparent cooperativity was reduced at shorter SL. These data emphasize that SL and/or myofilament lattice spacing modulation of the cross-bridge component of cardiac thin-filament activation contributes to the Frank-Starling mechanism. PMID:19413974

  9. Effect of oxidative stress on the expression of thin filament-associated proteins in gastric smooth muscle cells.

    PubMed

    Al-Shboul, Othman Abdullah; Mustafa, Ayman; Mohammad, Mukhallad; Al-Shehabat, Mustafa; Yousef, Asmaa; Al-Hashimi, Farah

    2014-09-01

    Thin filament-associated proteins such as calponin, caldesmon, and smoothelin are believed to regulate acto-myosin interaction and thus, muscle contraction. Oxidative stress has been found to affect the normal contractile behavior of smooth muscle and is involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the expression of smooth muscle contractile proteins. The aim of the current study is to investigate the effect of oxidative stress on the expression of thin filament-associated proteins in rat gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague-Dawley rats were used. Muscle cells were treated with hydrogen peroxide (H2O2) (500 μM) for 30 min or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) (1 mM) for 90 min to induce oxidative stress. Calponin, caldesmon, and smoothelin expressions were measured via specifically designed enzyme-linked immunosorbent assay. We found that exposure to exogenous H2O2 or incubation of dispersed gastric muscle cells with SIN-1 significantly increased the expression of calponin, caldesmon, and smoothelin proteins. In conclusion: oxidative stress increases the expression of thin filament-associated proteins in gastric smooth muscle, suggesting an important role in gastrointestinal motility disorders associated with oxidative stress.

  10. Electron microscopy and three-dimensional reconstruction of native thin filaments reveal species-specific differences in regulatory strand densities

    SciTech Connect

    Cammarato, Anthony; Craig, Roger; Lehman, William

    2010-01-01

    Throughout the animal kingdom striated muscle contraction is regulated by the thin filament troponin-tropomyosin complex. Homologous regulatory components are shared among vertebrate and arthropod muscles; however, unique protein extensions and/or components characterize the latter. The Troponin T (TnT) isoforms of Drosophila indirect flight and tarantula femur muscle for example contain distinct C-terminal extensions and are {approx}20% larger overall than their vertebrate counterpart. Using electron microscopy and three-dimensional helical reconstruction of native Drosophila, tarantula and frog muscle thin filaments we have identified species-specific differences in tropomyosin regulatory strand densities. The strands on the arthropod thin filaments were significantly larger in diameter than those from vertebrates, although not significantly different from each other. These findings reflect differences in the regulatory troponin-tropomyosin complex, which are likely due to the larger TnT molecules aligning and extending along much of the tropomyosin strands' length. Such an arrangement potentially alters the physical properties of the regulatory strands and may help establish contractile characteristics unique to certain arthropod muscles.

  11. Tropomyosin movement on F-actin during muscle activation explained by energy landscapes.

    PubMed

    Orzechowski, Marek; Moore, Jeffrey R; Fischer, Stefan; Lehman, William

    2014-03-01

    Muscle contraction is regulated by tropomyosin movement across the thin filament surface, which exposes or blocks myosin-binding sites on actin. Recent atomic structures of F-actin-tropomyosin have yielded the positions of tropomyosin on myosin-free and myosin-decorated actin. Here, the repositioning of α-tropomyosin between these locations on F-actin was systematically examined by optimizing the energy of the complex for a wide range of tropomyosin positions on F-actin. The resulting energy landscape provides a full-map of the F-actin surface preferred by tropomyosin, revealing a broad energy basin associated with the tropomyosin position that blocks myosin-binding. This is consistent with previously proposed low-energy oscillations of semi-rigid tropomyosin, necessary for shifting of tropomyosin following troponin-binding. In contrast, the landscape shows much less favorable energies when tropomyosin locates near its myosin-induced "open-state" position. This indicates that spontaneous movement of tropomyosin away from its energetic "ground-state" to the open-state is unlikely in absence of myosin. Instead, myosin-binding must drive tropomyosin toward the open-state to activate the thin filament. Additional energy landscapes were computed for disease-causing actin mutants that distort the topology of the actin-tropomyosin energy landscape, explaining their phenotypes. Thus, the computation of such energy landscapes offers a sensitive way to estimate the impact of mutations.

  12. Microtubules as Platforms for Assaying Actin Polymerization In Vivo

    PubMed Central

    Oelkers, J. Margit; Vinzenz, Marlene; Nemethova, Maria; Jacob, Sonja; Lai, Frank P. L.; Block, Jennifer; Szczodrak, Malgorzata; Kerkhoff, Eugen; Backert, Steffen; Schlüter, Kai; Stradal, Theresia E. B.; Small, J. Victor

    2011-01-01

    The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process. PMID:21603613

  13. Mechanics of F-actin characterized with microfabricated cantilevers.

    PubMed Central

    Liu, Xiumei; Pollack, Gerald H

    2002-01-01

    In this report we characterized the longitudinal elasticity of single actin filaments manipulated by novel silicon-nitride microfabricated levers. Single actin filaments were stretched from zero tension to maximal physiological tension, P(0). The obtained length-tension relation was nonlinear in the low-tension range (0-50 pN) with a resultant strain of approximately 0.4-0.6% and then became linear at moderate to high tensions (approximately 50-230 pN). In this region, the stretching stiffness of a single rhodamine-phalloidin-labeled, 1-microm-long F-actin is 34.5 +/- 3.5 pN/nm. Such a length-tension relation could be characterized by an entropic-enthalpic worm-like chain model, which ascribes most of the energy consumed in the nonlinear portion to overcoming thermal undulations arising from the filament's interaction with surrounding solution and the linear portion to the intrinsic stretching elasticity. By fitting the experimental data with such a worm-like chain model, an estimation of persistence length of approximately 8.75 microm was derived. These results suggest that F-actin is more compliant than previously thought and that thin filament compliance may account for a substantial fraction of the sarcomere's elasticity. PMID:12414703

  14. Differential binding of tropomyosin isoforms to actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester and fluorescein-5-isothiocyanate.

    PubMed

    Skórzewski, Radosław; Robaszkiewicz, Katarzyna; Jarzebińska, Justyna; Suder, Piotr; Silberring, Jerzy; Moraczewska, Joanna

    2009-11-01

    Differential interactions of tropomyosin (TM) isoforms with actin can be important for determination of the thin filament functions. A mechanism of tropomyosin binding to actin was studied by comparing interactions of five alphaTM isoforms with actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and with fluorescein-5-isothiocyanate (FITC). MBS attachment sites were revealed with mass spectrometry methods. We found that the predominant actin fraction was cross-linked by MBS within subdomain 3. A smaller fraction of the modified actin was cross-linked within subdomain 2 and between subdomains 2 and 1. Moreover, investigated actins carried single labels in subdomains 1, 2, and 3. Such extensive modification caused a large decrease in actin affinity for skeletal and smooth muscle tropomyosins, nonmuscle TM2, and chimeric TM1b9a. In contrast, binding of nonmuscle isoform TM5a was less affected. Isoform's affinity for actin modified in subdomain 2 by binding of FITC to Lys61 was intermediate between the affinity for native actin and MBS-modified actin except for TM5a, which bound to FITC-actin with similar affinity as to actin modified with MBS. The analysis of binding curves according to the McGhee-von Hippel model revealed that binding to an isolated site, as well as cooperativity of binding to a contiguous site, was affected by both actin modifications in a TM isoform-specific manner.

  15. Arabidopsis Villins Promote Actin Turnover at Pollen Tube Tips and Facilitate the Construction of Actin Collars[W

    PubMed Central

    Qu, Xiaolu; Zhang, Hua; Xie, Yurong; Wang, Juan; Chen, Naizhi; Huang, Shanjin

    2013-01-01

    Apical actin filaments are crucial for pollen tube tip growth. However, the specific dynamic changes and regulatory mechanisms associated with actin filaments in the apical region remain largely unknown. Here, we have investigated the quantitative dynamic parameters that underlie actin filament growth and disappearance in the apical regions of pollen tubes and identified villin as the major player that drives rapid turnover of actin filaments in this region. Downregulation of Arabidopsis thaliana VILLIN2 (VLN2) and VLN5 led to accumulation of actin filaments at the pollen tube apex. Careful analysis of single filament dynamics showed that the severing frequency significantly decreased, and the lifetime significantly increased in vln2 vln5 pollen tubes. These results indicate that villin-mediated severing is critical for turnover and departure of actin filaments originating in the apical region. Consequently, the construction of actin collars was affected in vln2 vln5 pollen tubes. In addition to the decrease in severing frequency, actin filaments also became wavy and buckled in the apical cytoplasm of vln2 vln5 pollen tubes. These results suggest that villin confers rigidity upon actin filaments. Furthermore, an observed decrease in skewness of actin filaments in the subapical region of vln2 vln5 pollen tubes suggests that villin-mediated bundling activity may also play a role in the construction of actin collars. Thus, our data suggest that villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars. PMID:23715472

  16. Cardiac myosin-binding protein C decorates F-actin: Implications for cardiac function

    PubMed Central

    Whitten, Andrew E.; Jeffries, Cy M.; Harris, Samantha P.; Trewhella, Jill

    2008-01-01

    Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin. PMID:19011110

  17. Bacterial Actins and Their Interactors.

    PubMed

    Gayathri, Pananghat

    2017-01-01

    Bacterial actins polymerize in the presence of nucleotide (preferably ATP), form a common arrangement of monomeric interfaces within a protofilament, and undergo ATP hydrolysis-dependent change in stability of the filament-all of which contribute to performing their respective functions. The relative stability of the filament in the ADP-bound form compared to that of ATP and the rate of addition of monomers at the two ends decide the filament dynamics. One of the major differences between eukaryotic actin and bacterial actins is the variety in protofilament arrangements and dynamics exhibited by the latter. The filament structure and the polymerization dynamics enable them to perform various functions such as shape determination in rod-shaped bacteria (MreB), cell division (FtsA), plasmid segregation (ParM family of actin-like proteins), and organelle positioning (MamK). Though the architecture and dynamics of a few representative filaments have been studied, information on the effect of interacting partners on bacterial actin filament dynamics is not very well known. The chapter reviews some of the structural and functional aspects of bacterial actins, with special focus on the effect that interacting partners exert on the dynamics of bacterial actins, and how these assist them to carry out the functions within the bacterial cell.

  18. Regulatory mechanism of length-dependent activation in skinned porcine ventricular muscle: role of thin filament cooperative activation in the Frank-Starling relation.

    PubMed

    Terui, Takako; Shimamoto, Yuta; Yamane, Mitsunori; Kobirumaki, Fuyu; Ohtsuki, Iwao; Ishiwata, Shin'ichi; Kurihara, Satoshi; Fukuda, Norio

    2010-10-01

    Cardiac sarcomeres produce greater active force in response to stretch, forming the basis of the Frank-Starling mechanism of the heart. The purpose of this study was to provide the systematic understanding of length-dependent activation by investigating experimentally and mathematically how the thin filament "on-off" switching mechanism is involved in its regulation. Porcine left ventricular muscles were skinned, and force measurements were performed at short (1.9 µm) and long (2.3 µm) sarcomere lengths. We found that 3 mM MgADP increased Ca(2+) sensitivity of force and the rate of rise of active force, consistent with the increase in thin filament cooperative activation. MgADP attenuated length-dependent activation with and without thin filament reconstitution with the fast skeletal troponin complex (sTn). Conversely, 20 mM of inorganic phosphate (Pi) decreased Ca(2+) sensitivity of force and the rate of rise of active force, consistent with the decrease in thin filament cooperative activation. Pi enhanced length-dependent activation with and without sTn reconstitution. Linear regression analysis revealed that the magnitude of length-dependent activation was inversely correlated with the rate of rise of active force. These results were quantitatively simulated by a model that incorporates the Ca(2+)-dependent on-off switching of the thin filament state and interfilament lattice spacing modulation. Our model analysis revealed that the cooperativity of the thin filament on-off switching, but not the Ca(2+)-binding ability, determines the magnitude of the Frank-Starling effect. These findings demonstrate that the Frank-Starling relation is strongly influenced by thin filament cooperative activation.

  19. Regulatory mechanism of length-dependent activation in skinned porcine ventricular muscle: role of thin filament cooperative activation in the Frank-Starling relation

    PubMed Central

    Terui, Takako; Shimamoto, Yuta; Yamane, Mitsunori; Kobirumaki, Fuyu; Ohtsuki, Iwao; Ishiwata, Shin’ichi; Kurihara, Satoshi

    2010-01-01

    Cardiac sarcomeres produce greater active force in response to stretch, forming the basis of the Frank-Starling mechanism of the heart. The purpose of this study was to provide the systematic understanding of length-dependent activation by investigating experimentally and mathematically how the thin filament “on–off” switching mechanism is involved in its regulation. Porcine left ventricular muscles were skinned, and force measurements were performed at short (1.9 µm) and long (2.3 µm) sarcomere lengths. We found that 3 mM MgADP increased Ca2+ sensitivity of force and the rate of rise of active force, consistent with the increase in thin filament cooperative activation. MgADP attenuated length-dependent activation with and without thin filament reconstitution with the fast skeletal troponin complex (sTn). Conversely, 20 mM of inorganic phosphate (Pi) decreased Ca2+ sensitivity of force and the rate of rise of active force, consistent with the decrease in thin filament cooperative activation. Pi enhanced length-dependent activation with and without sTn reconstitution. Linear regression analysis revealed that the magnitude of length-dependent activation was inversely correlated with the rate of rise of active force. These results were quantitatively simulated by a model that incorporates the Ca2+-dependent on–off switching of the thin filament state and interfilament lattice spacing modulation. Our model analysis revealed that the cooperativity of the thin filament on–off switching, but not the Ca2+-binding ability, determines the magnitude of the Frank-Starling effect. These findings demonstrate that the Frank-Starling relation is strongly influenced by thin filament cooperative activation. PMID:20876361

  20. Polycation induced actin bundles.

    PubMed

    Muhlrad, Andras; Grintsevich, Elena E; Reisler, Emil

    2011-04-01

    Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.

  1. Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments

    NASA Astrophysics Data System (ADS)

    Hariadi, R. F.; Sommese, R. F.; Adhikari, A. S.; Taylor, R. E.; Sutton, S.; Spudich, J. A.; Sivaramakrishnan, S.

    2015-08-01

    The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

  2. During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

    PubMed Central

    Cohen, Shenhav; Brault, Jeffrey J.; Gygi, Steven P.; Glass, David J.; Valenzuela, David M.; Gartner, Carlos; Latres, Esther

    2009-01-01

    Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1. PMID:19506036

  3. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  4. Direct tests of muscle cross-bridge theories: predictions of a Brownian dumbbell model for position-dependent cross-bridge lifetimes and step sizes with an optically trapped actin filament.

    PubMed Central

    Smith, D A

    1998-01-01

    Force and displacement events from a single myosin molecule interacting with an actin filament suspended between optically trapped beads (Finer, J. T., R. M. Simmons, and J. A. Spudich. 1994. Nature. 368:113-119) can be interpreted in terms of a generalized cross-bridge model that includes the effects of Brownian forces on the beads. Steady-state distributions of force and displacement can be obtained directly from a generalized Smoluchowski equation for Brownian motion of the actin-bead "dumbbell," and time series from Monte Carlo simulations of the corresponding Langevin equation. When the frequency spectrum of Brownian motion extends beyond cross-bridge transition rates, the inverse mean lifetimes of force/displacement pulses are given by cross-bridge rate constants averaged over a Boltzmann distribution of Brownian noise. These averaged rate constants reflect the strain-dependence of the rate constants for the stationary filament, most faithfully at high trap stiffness. Hence, measurements of the lifetimes and displacements of single events as a function of the resting position of the dumbbell can provide a direct test of different cross-bridge theories of muscle contraction. Quantitative demonstrations are given for Huxley models with 1) faster binding or 2) slower dissociation at positive cross-bridge strain. Predictions for other models can be inferred from the averaging procedure. PMID:9826619

  5. Ca++-sensitizing mutations in troponin, P(i), and 2-deoxyATP alter the depressive effect of acidosis on regulated thin-filament velocity.

    PubMed

    Longyear, Thomas J; Turner, Matthew A; Davis, Jonathan P; Lopez, Joseph; Biesiadecki, Brandon; Debold, Edward P

    2014-05-01

    Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, resulting in fatigue. The decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains poorly understood. We sought to gain novel insight into the decrease in velocity by determining whether the depressive effect of acidosis could be altered by 1) introducing Ca(++)-sensitizing mutations into troponin (Tn) or 2) by agents that directly affect myosin function, including inorganic phosphate (Pi) and 2-deoxy-ATP (dATP) in an in vitro motility assay. Acidosis reduced regulated thin-filament velocity (VRTF) at both maximal and submaximal Ca(++) levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn (TnI) and a Ca(++)-sensitizing mutation in the Ca(++)-binding subunit of Tn (TnC) increased VRTF at submaximal Ca(++) under acidic conditions but had no effect on VRTF at maximal Ca(++) levels. In contrast, both Pi and replacement of ATP with dATP reversed much of the acidosis-induced depression of VRTF at saturating Ca(++). Interestingly, despite producing similar magnitude increases in VRTF, the combined effects of Pi and dATP were additive, suggesting different underlying mechanisms of action. These findings suggest that acidosis depresses velocity by slowing the detachment rate from actin but also by possibly slowing the attachment rate.

  6. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex*

    PubMed Central

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-01-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and the trans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H+-translocating ATPase (V-ATPase), whose V1 domain subunits B and C bind actin. We have generated a GFP-tagged subunit B2 construct (GFP-B2) that is incorporated into the V1 domain, which in turn is coupled to the V0 sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0 domains, which entails subunit B2 translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunits B2 and C1 and actin were detected. In addition, Golgi membrane lipid order disruption by d-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0 domains of V-ATPase through the binding of microfilaments to subunits B and C and preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH. PMID:26872971

  7. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex.

    PubMed

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-04-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH.

  8. Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells.

    PubMed Central

    Lang, T; Wacker, I; Wunderlich, I; Rohrbach, A; Giese, G; Soldati, T; Almers, W

    2000-01-01

    In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion. PMID:10827968

  9. In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins.

    PubMed

    Zimmermann, Dennis; Morganthaler, Alisha N; Kovar, David R; Suarez, Cristian

    2016-01-01

    Characterizing the biochemical and biophysical properties of purified proteins is critical to understand the underlying molecular mechanisms that facilitate complicated cellular processes such as cytokinesis. Here we outline in vitro assays to investigate the effects of cytokinesis actin-binding proteins on actin filament dynamics and organization. We describe (1) multicolor single-molecule TIRF microscopy actin assembly assays, (2) "bulk" pyrene actin assembly/disassembly assays, and (3) "bulk" sedimentation actin filament binding and bundling assays.

  10. Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase

    PubMed Central

    1995-01-01

    Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti- phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas

  11. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  12. Human congenital myopathy actin mutants cause myopathy and alter Z-disc structure in Drosophila flight muscle.

    PubMed

    Sevdali, Maria; Kumar, Vikash; Peckham, Michelle; Sparrow, John

    2013-03-01

    Over 190 mutations in the human skeletal muscle α-actin gene, ACTA1 cause congenital actin myopathies. We transgenically expressed six different mutant actins, G15R, I136M, D154N, V163L, V163M and D292V in Drosophila indirect flight muscles and investigated their effects in flies that express one wild type and one mutant actin copy. All the flies were flightless, and the IFMs showed incomplete Z-discs, disorganised actin filaments and 'zebra bodies'. No differences in levels of sarcomeric protein expression were observed, but tropomodulin staining was somewhat disrupted in D164N, V163L, G15R and V163M heterozygotes. A single copy of D292V mutant actin rescued the hypercontractile phenotypes caused by TnI and TnT mutants, suggesting that the D292V mutation interferes with thin filament regulation. Our results show that expression of actin mutations homologous to those in humans in the indirect flight muscles of Drosophila disrupt sarcomere organisation, with somewhat similar phenotypes to those observed in humans. Using Drosophila to study actin mutations may help aid our understanding of congential myopathies caused by actin mutations.

  13. Spatial control of the actin cytoskeleton in Drosophila epithelial cells.

    PubMed

    Baum, B; Perrimon, N

    2001-10-01

    The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.

  14. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  15. The effects of collapsing factors on F-actin content and microtubule distribution of Helisoma growth cones.

    PubMed

    Torreano, Paul J; Waterman-Storer, Clare M; Cohan, Christopher S

    2005-03-01

    Growth cone collapsing factors induce growth cone collapse or repulsive growth cone turning by interacting with membrane receptors that induce alterations in the growth cone cytoskeleton. A common change induced by collapsing factors in the cytoskeleton of the peripheral domain, the thin lamellopodial area of growth cones, is a decline in the number of radially aligned F-actin bundles that form the core of filopodia. The present study examined whether ML-7, a myosin light chain kinase inhibitor, serotonin, a neurotransmitter and TPA, an activator of protein kinase C, which induce growth cone collapse of Helisoma growth cones, depolymerized or debundled F-actin. We report that these collapsing factors had different effects. ML-7 induced F-actin reorganization consistent with debundling whereas serotonin and TPA predominately depolymerized and possibly debundled F-actin. Additionally, these collapsing factors induced the formation of a dense actin-ring around the central domain, the thicker proximal area of growth cones [Zhou and Cohan, 2001: J. Cell Biol. 153:1071-1083]. The formation of the actin-ring occurred subsequent to the loss of actin bundles. The ML-7-induced actin-ring was found to inhibit microtubule extension into the P-domain. Thus, ML-7, serotonin, and TPA induce growth cone collapse associated with a decline in radially aligned F-actin bundles through at least two mechanisms involving debundling of actin filaments and/or actin depolymerization.

  16. Structural Differences Explain Diverse Functions of Plasmodium Actins

    PubMed Central

    Vahokoski, Juha; Martinez, Silvia Muñico; Ignatev, Alexander; Lepper, Simone; Frischknecht, Friedrich; Sidén-Kiamos, Inga; Sachse, Carsten; Kursula, Inari

    2014-01-01

    Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties. PMID:24743229

  17. Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles.

    PubMed Central

    Miyata, H; Hotani, H

    1992-01-01

    Spherical giant liposomes that had encapsulated skeletal-muscle G-actin were made by swelling a dried lipid mixture of dimyristoyl phosphatidylcholine/cardiolipin, 1:1 (wt/wt), in a solution of G-actin/CaCl2 at 0 degree C. Polymerization of the encapsulated G-actin into actin filaments was achieved by raising the temperature to 30 degrees C. We observed the subsequent shape changes of the liposomes by dark-field and differential interference-contrast light microscopy. After approximately 40 min, which was required for completion of actin polymerization, two shapes of liposome were evident: dumbbell and disk. Elongation of the dumbbell-shaped liposomes was concomitant with actin polymerization. Polarization microscopy showed that actin filaments formed thick bundles in the liposomes and that these filaments lay contiguous to the periphery of the liposome. Localization of actin filaments in the liposomes was confirmed by observation of rhodamine phalloidin-conjugated actin filaments by fluorescence microscopy. Both dumbbell- and disk-shaped liposomes were rigid and kept their shapes as far as actin filaments were stabilized. In contrast, liposomes containing bovine serum albumin were fragile, and their shapes continually fluctuated from Brownian motion, indicating that the actin bundles served as mechanical support for the liposome shapes. Images PMID:1454846

  18. Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

    SciTech Connect

    Aihara, Tomoki . E-mail: arata@bio.sci.osaka-u.ac.jp

    2006-02-10

    We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca{sup 2+}-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca{sup 2+} state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time {tau} = 1.9 ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca{sup 2+} state showed that Cys133 existed on a stable segment ({tau} = 4.8 ns) held by TnC. Spectra of reconstituted thin filaments (-Ca{sup 2+} state) revealed that slow mobility ({tau} = 45 ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca{sup 2+} concentrations.

  19. Changes in morphology of actin filaments and expression of alkaline phosphatase at 3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds.

    PubMed

    Goncharenko, A V; Malyuchenko, N V; Moisenovich, A M; Kotlyarova, M S; Arkhipova, A Yu; Kon'kov, A S; Agapov, I I; Molochkov, A V; Moisenovich, M M; Kirpichnikov, M P

    2016-09-01

    3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds leads to an increase in the expression of alkaline phosphatase, an early marker of bone formation. Increased expression is associated with the actin cytoskeleton reorganization under the influence of 3D cultivation and osteogenic calcium phosphate component of the microcarrier.

  20. Solid friction between soft filaments.

    PubMed

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A W C; Vitelli, Vincenzo; Mahadevan, L; Dogic, Zvonimir

    2015-06-01

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments' overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes's drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. Our findings demonstrate how altering a filament's elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.

  1. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    PubMed Central

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. PMID:21444821

  2. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

    PubMed

    Paredez, Alexander R; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C; Wang, Chung-Ju Rachel; Cande, W Z

    2011-04-12

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

  3. Lifeact: a versatile marker to visualize F-actin.

    PubMed

    Riedl, Julia; Crevenna, Alvaro H; Kessenbrock, Kai; Yu, Jerry Haochen; Neukirchen, Dorothee; Bista, Michal; Bradke, Frank; Jenne, Dieter; Holak, Tad A; Werb, Zena; Sixt, Michael; Wedlich-Soldner, Roland

    2008-07-01

    Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

  4. An ecto-protein tyrosine phosphatase of Entamoeba histolytica induces cellular detachment by disruption of actin filaments in HeLa cells.

    PubMed

    Anaya-Ruiz, M; Pérez-Santos, J L M; Talamás-Rohana, P

    2003-07-01

    Actin cytoskeleton disruption in host cells has been demonstrated for PTPases from pathogenic microorganisms. In this work, we analysed whether the secreted acid phosphatase from Entamoeba histolytica has phosphotyrosine phosphatase activity and the possibility that this activity may participate in damaging host cells. The secreted acid phosphatase of E. histolytica, which catalyses p-nitrophenyl phosphate hydrolysis at acid pH values, was found to have phosphotyrosine phosphatase activity. The enzymatic properties of phosphotyrosine phosphatase and acid phosphatase were virtually identical and included: Km values of 10 x 10(-4) M, no requirement for divalent cations, and sensitivity to molybdate, vanadate, and tungstate. The phosphotyrosyl phosphatase activity caused significant levels of cell rounding and detachment correlating with disruption of the actin stress fibres in HeLa cells. Thus, our data suggest that secreted phosphotyrosine phosphatase could play a cytotoxic role during amoebic infection.

  5. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  6. [Conformational changes of actin induced by strong or weak myosin subfragment-1 binding].

    PubMed

    Dedova, I V; Avrova, S V; Vikhoreva, N N; Vikhorev, R G; Hazlett, T L; Van der Meer, W; Dos Remedios, C G; Borovikov, Iu S

    2004-01-01

    Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.

  7. Actin dynamics: old friends with new stories.

    PubMed

    Staiger, Christopher J; Blanchoin, Laurent

    2006-12-01

    Actin dynamics, or the rapid turnover of actin filaments, play a central role in numerous cellular processes. A large and diverse cast of characters, accessory proteins known as actin-binding proteins, modulate actin dynamics. They do this by binding to the monomer pool, interacting with the side and ends of filaments, creating breaks along a filament, and generating new filaments de novo. Recent biochemical and single-filament imaging analyses of several conserved classes of plant actin-binding proteins reveal unusual and unexpected properties. Examples that are highlighted in this review include: an abundant monomer-binding protein that catalyzes nucleotide exchange; a barbed-end capping protein that is dissociated from filament ends by the signaling lipid, phosphatidic acid; a villin-like bundling protein that lacks all Ca(2+)-regulated activities; and a formin family member that is non-processive and is sufficient to generate actin filament bundles. These and other stories motivate a careful description of the properties of plant proteins in vitro as a prelude to greater insight into the molecular mechanism(s) underlying the regulation of actin dynamics in vivo.

  8. Nucleus-associated actin in Amoeba proteus.

    PubMed

    Berdieva, Mariia; Bogolyubov, Dmitry; Podlipaeva, Yuliya; Goodkov, Andrew

    2016-10-01

    The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.

  9. In inflammatory reactive astrocytes co-cultured with brain endothelial cells nicotine-evoked Ca(2+) transients are attenuated due to interleukin-1beta release and rearrangement of actin filaments.

    PubMed

    Delbro, D; Westerlund, A; Björklund, U; Hansson, E

    2009-03-17

    The aim of this study was to investigate whether nicotine acetylcholine receptors (nAChRs) are expressed in a more pronounced way in astrocytes co-cultured with microvascular endothelial cells from adult rat brain, compared with monocultured astrocytes, as a sign of a more developed signal transduction system. Also investigated was whether nicotine plays a role in the control of neuroinflammatory reactivity in astrocytes. Ca(2+) imaging experiments were performed using cells loaded with the Ca(2+) indicator Fura-2/AM. Co-cultured astrocytes responded to lower concentrations of nicotine than did monocultured astrocytes, indicating that they are more sensitive to nicotine. Co-cultured astrocytes also expressed a higher selectivity for alpha7nAChR and alpha4/beta2 subunits and evoked higher Ca(2+) transients compared with monocultured astrocytes. The Ca(2+) transients referred to are activators of Ca(2+)-induced Ca(2+) release from intracellular stores, both IP(3) and ryanodine, triggered by influx through receptor channels. The nicotine-induced Ca(2+) transients were attenuated after incubation with the inflammatory mediator lipopolysaccharide (LPS), but were not attenuated after incubation with the pain-transmitting peptides substance P and calcitonin-gene-related peptide, nor with the infection and inflammation stress mediator, leptin. Furthermore, LPS-induced release of interleukin-1beta (IL-1beta) measured by enzyme-linked immunosorbent assay (ELISA) was more pronounced in co-cultured versus monocultured astrocytes. Incubation with both LPS and IL-1beta further attenuated nicotine-induced Ca(2+) response. We also found that LPS and IL-1beta induced rearrangement of the F-actin filaments, as measured with an Alexa488-conjugated phalloidin probe. The rearrangements consisted of increases in ring formations and a more dispersed appearance of the filaments. These results indicate that there is a connection between a dysfunction of nicotine Ca(2+) signaling in

  10. Structure of the Rigor Actin-Tropomyosin-Myosin Complex

    PubMed Central

    Behrmann, Elmar; Müller, Mirco; Penczek, Pawel A.; Mannherz, Hans Georg; Manstein, Dietmar J.; Raunser, Stefan

    2014-01-01

    The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. Myosin converts the chemical energy stored in ATP into force and movement along actin filaments. Myosin binding to actin induces conformational changes that are coupled to the nucleotide-binding pocket and amplified by a specialized region of the motor domain for efficient force generation. Tropomyosin plays a key role in regulating the productive interaction between myosins and actin. Here, we report the 8 Å resolution structure of the actin-tropomyosin-myosin complex determined by cryo electron microscopy. The pseudo-atomic model of the complex obtained from fitting crystal structures into the map defines the large actin-myosin-tropomyosin interface and the molecular interactions between the proteins in detail and allows us to propose a structural model for tropomyosin dependent myosin binding to actin and actin-induced nucleotide release from myosin. PMID:22817895

  11. Using baculovirus/insect cell expressed recombinant actin to study the molecular pathogenesis of HCM caused by actin mutation A331P

    PubMed Central

    Bai, Fan; Caster, Hannah M.; Rubenstein, Peter A.; Dawson, John F.; Kawai, Masataka

    2014-01-01

    Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca2+], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25°C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75±0.06 T0, N=11; BVC: 0.73±0.07 T0, N=12, where T0 is tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85±0.06 T0 (N=11) compared to 0.98±0.04 T0 (N=12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca2+ sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69±0.01, N=10; WT: 5.69±0.02, N=11; BVC: 5.68±0.02, N=12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers, and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55±0.03 T0, N=13) and in their presence (0.65±0.02 T0, N=13) compared to those of the WT (0.75±0

  12. Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog.

    PubMed

    Reconditi, Massimo; Brunello, Elisabetta; Fusi, Luca; Linari, Marco; Martinez, Manuel Fernandez; Lombardi, Vincenzo; Irving, Malcolm; Piazzesi, Gabriella

    2014-03-01

    X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments.

  13. Nitric oxide modulates the influx of extracellular Ca2+ and actin filament organization during cell wall construction in Pinus bungeana pollen tubes.

    PubMed

    Wang, Yuhua; Chen, Tong; Zhang, Chunyang; Hao, Huaiqing; Liu, Peng; Zheng, Maozhong; Baluška, František; Šamaj, Jozef; Lin, Jinxing

    2009-06-01

    Nitric oxide (NO) plays a key role in many physiological processes in plants, including pollen tube growth. Here, effects of NO on extracellular Ca(2+) flux and microfilaments during cell wall construction in Pinus bungeana pollen tubes were investigated. Extracellular Ca(2+) influx, the intracellular Ca(2+) gradient, patterns of actin organization, vesicle trafficking and cell wall deposition upon treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP), the NO synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NNA) or the NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were analyzed. SNAP enhanced pollen tube growth in a dose-dependent manner, while L-NNA and cPTIO inhibited NO production and arrested pollen tube growth. Noninvasive detection and microinjection of a Ca(2+) indicator revealed that SNAP promoted extracellular Ca(2+) influx and increased the steepness of the tip-focused Ca(2+) gradient, while cPTIO and L-NNA had the opposite effect. Fluorescence labeling indicated that SNAP, cPTIO and L-NNA altered actin organization, which subsequently affected vesicle trafficking. Finally, the configuration and/or distribution of cell wall components such as pectins and callose were significantly altered in response to L-NNA. Fourier transform infrared (FTIR) microspectroscopy confirmed the changes in the chemical composition of walls. Our results indicate that NO affects the configuration and distribution of cell wall components in pollen tubes by altering extracellular Ca(2+) influx and F-actin organization.

  14. Labeling F-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones.

    PubMed

    Marsick, Bonnie M; Letourneau, Paul C

    2011-03-17

    The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant. Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells. When rhodamine-actin is added at a concentration above the critical concentration

  15. Dual conical conducting filament model in resistance switching TiO2 thin films.

    PubMed

    Kim, Kyung Min; Park, Tae Hyung; Hwang, Cheol Seong

    2015-01-19

    The resetting behaviors of Pt/TiO2/Pt resistive switching (RS) cell in unipolar RS operations were studied in detail through an experiment and by modeling. The experiment showed that the apparently highly arbitrary resetting current-voltage (I-V) curves could be grouped into three types: normal, delayed, and abnormal behaviors. A dual conical conducting filament (CF) model was conceived, and their electrothermal behaviors were analytically described from the heat-balance and charge-transport equations. The almost spontaneous resetting behavior of the normal reset could be easily understood from the mutually constructive interference effect between the Joule heating and temperature-dependent resistance effect along the CF. The delayed reset could be explained by the time-dependent increase in the reset voltage during the rest process, which was most probably induced in the more conical-shaped CF. The abnormal reset could be understood from the temporal transfer of oxygen ions near the kink positions of the two different-diameter portions of the more cylindrical CFs, which temporally decreases the overall resistance immediately prior for the actual reset to occur. The accuracy of the dual conical CF model was further confirmed by adopting a more thorough electrothermal simulation package, COMSOL.

  16. Dual Conical Conducting Filament Model in Resistance Switching TiO2 Thin Films

    NASA Astrophysics Data System (ADS)

    Kim, Kyung Min; Park, Tae Hyung; Hwang, Cheol Seong

    2015-01-01

    The resetting behaviors of Pt/TiO2/Pt resistive switching (RS) cell in unipolar RS operations were studied in detail through an experiment and by modeling. The experiment showed that the apparently highly arbitrary resetting current-voltage (I-V) curves could be grouped into three types: normal, delayed, and abnormal behaviors. A dual conical conducting filament (CF) model was conceived, and their electrothermal behaviors were analytically described from the heat-balance and charge-transport equations. The almost spontaneous resetting behavior of the normal reset could be easily understood from the mutually constructive interference effect between the Joule heating and temperature-dependent resistance effect along the CF. The delayed reset could be explained by the time-dependent increase in the reset voltage during the rest process, which was most probably induced in the more conical-shaped CF. The abnormal reset could be understood from the temporal transfer of oxygen ions near the kink positions of the two different-diameter portions of the more cylindrical CFs, which temporally decreases the overall resistance immediately prior for the actual reset to occur. The accuracy of the dual conical CF model was further confirmed by adopting a more thorough electrothermal simulation package, COMSOL.

  17. CLIC5 Stabilizes Membrane-Actin Filament Linkages at the Base of Hair Cell Stereocilia in a Molecular Complex with Radixin, Taperin, and Myosin VI

    PubMed Central

    Salles, Felipe T.; Andrade, Leonardo R.; Tanda, Soichi; Grati, M’hamed; Plona, Kathleen L.; Gagnon, Leona H.; Johnson, Kenneth R.; Kachar, Bechara; Berryman, Mark A.

    2015-01-01

    Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of stereocilia, were mislocalized in fused stereocilia of jbg mice. TPRQ and RDX were dispersed even prior to stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of stereocilia. PMID:24285636

  18. X-ray recordings reveal how a human disease-linked skeletal muscle α-actin mutation leads to contractile dysfunction.

    PubMed

    Ochala, Julien; Ravenscroft, Gianina; McNamara, Elyshia; Nowak, Kristen J; Iwamoto, Hiroyuki

    2015-12-01

    In humans, mutant skeletal muscle α-actin proteins are associated with contractile dysfunction, skeletal muscle weakness and a wide range of primarily skeletal muscle diseases. Despite this knowledge, the exact molecular mechanisms triggering the contractile dysfunction remain unknown. Here, we aimed to unravel these. Hence, we used a transgenic mouse model expressing a well-described D286G mutant skeletal muscle α-actin protein and recapitulating the human condition of contractile deregulation and severe skeletal muscle weakness. We then recorded and analyzed the small-angle X-ray diffraction patterns of isolated membrane-permeabilized myofibers. Results showed that upon addition of Ca(2+), the intensity changes of the second (1/19 nm(-1)) and sixth (1/5.9 nm(-1)) actin layer lines and of the first myosin meridional reflection (1/14.3 nm(-1)) were disrupted when the thin-thick filament overlap was optimal (sarcomere length of 2.5-2.6 μm). However these reflections were normal when the thin and thick filaments were not interacting (sarcomere length>3.6 μm). These findings demonstrate, for the first time, that the replacement of just one amino acid in the skeletal muscle α-actin protein partly prevents actin conformational changes during activation, disrupting the strong binding of myosin molecules. This leads to a limited myosin-related tropomyosin movement over the thin filaments, further affecting the amount of cross-bridges, explaining the contractile dysfunction.

  19. Applicability of the hodograph method for the problem of long-scale nonlinear dynamics of a thin vortex filament near a flat boundary.

    PubMed

    Ruban, V P

    2003-06-01

    Hamiltonian dynamics of a thin vortex filament in an ideal incompressible fluid near a flat fixed boundary is considered under the conditions that at any point of the curve, determining the shape of the filament, the angle between tangent vector and the boundary plane is small. Also the distance from a point on the curve to the plane is small in comparison with the curvature radius. The dynamics is shown to be effectively described by a nonlinear system of two (1+1)-dimensional partial differential equations. The hodograph transformation reduces this system to a single linear differential equation of the second order with separable variables. Simple solutions of the linear equation are investigated for real values of spectral parameter lambda, when the filament projection on the boundary plane has shape of a two-branch spiral or a smoothed angle, depending on the sign of lambda.

  20. Actin dynamics and cofilin-actin rods in Alzheimer disease

    PubMed Central

    Bamburg, James R.; Bernstein, Barbara W.

    2017-01-01

    Cytoskeletal abnormalities and synaptic loss, typical of both familial and sporadic Alzheimer disease (AD), are induced by diverse stresses such as neuroinflammation, oxidative stress, and energetic stress, each of which may be initiated or enhanced by proinflammatory cytokines or amyloid-β (Aβ) peptides. Extracellular Aβ-containing plaques and intracellular phospho-tau-containing neurofibrillary tangles are postmortem pathologies required to confirm AD and have been the focus of most studies. However, AD brain, but not normal brain, also have increased levels of cytoplasmic rod-shaped bundles of filaments composed of ADF/cofilin-actin in a 1:1 complex (rods). Cofilin, the major ADF/cofilin isoform in mammalian neurons, severs actin filaments at low cofilin/actin ratios and stabilizes filaments at high cofilin/actin ratios. It binds cooperatively to ADP-actin subunits in F-actin. Cofilin is activated by dephosphorylation and may be oxidized in stressed neurons to form disulfide-linked dimers, required for bundling cofilin-actin filaments into stable rods. Rods form within neurites causing synaptic dysfunction by sequestering cofilin, disrupting normal actin dynamics, blocking transport, and exacerbating mitochondrial membrane potential loss. Aβ and proinflammatory cytokines induce rods through a cellular prion protein-dependent activation of NADPH oxidase and production of reactive oxygen species. Here we review recent advances in our understanding of cofilin biochemistry, rod formation, and the development of cognitive deficits. We will then discuss rod formation as a molecular pathway for synapse loss that may be common between all three prominent current AD hypotheses, thus making rods an attractive therapeutic target. PMID:26873625

  1. Interaction of calponin with actin and its functional implications.

    PubMed Central

    Kołakowski, J; Makuch, R; Stepkowski, D; Dabrowska, R

    1995-01-01

    Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP. Images Figure 2 PMID:7864810

  2. Dual pools of actin at presynaptic terminals.

    PubMed

    Bleckert, Adam; Photowala, Huzefa; Alford, Simon

    2012-06-01

    We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

  3. Fabrication and superconducting properties of multifilamentary MgB2 conductors for AC purposes: twisted tapes and wires with very thin filaments

    NASA Astrophysics Data System (ADS)

    Malagoli, A.; Bernini, C.; Braccini, V.; Fanciulli, C.; Romano, G.; Vignolo, M.

    2009-10-01

    In order to use MgB2 conductors for AC applications, research and development efforts have to be carried out on their architecture and sheath material to minimize the AC losses. This paper will present the fabrication and characterization of two types of ex situ powder-in-tube processed pure MgB2 conductors with properties making them good candidates for AC industrial applications: a multifilamentary tape with 12 filaments with a twisting pitch down to 17 mm and a 361-filament wire with an average single-filament size of about 30 µm. Concerning the twisted tapes we will present values of critical current density of about 105 A cm-2 at 4.2 K and 2 T and we will show that it is possible to achieve a proper compromise between the tape size, the twisting pitch length and the critical current density to face a reduction of the critical current density as a consequence of the strain on the filaments. Concerning the 361-multifilamentary wire we will show appreciable values of critical current density of about 5 × 104 A cm-2 at 4.2 K and 2.5 T which, together with the advantages given by the high number of very thin filaments and the non-magnetic matrix, could justify their employment.

  4. Fast two-dimensional bubble analysis of biopolymer filamentous networks pore size from confocal microscopy thin data stacks.

    PubMed

    Molteni, Matteo; Magatti, Davide; Cardinali, Barbara; Rocco, Mattia; Ferri, Fabio

    2013-03-05

    The average pore size ξ0 of filamentous networks assembled from biological macromolecules is one of the most important physical parameters affecting their biological functions. Modern optical methods, such as confocal microscopy, can noninvasively image such networks, but extracting a quantitative estimate of ξ0 is a nontrivial task. We present here a fast and simple method based on a two-dimensional bubble approach, which works by analyzing one by one the (thresholded) images of a series of three-dimensional thin data stacks. No skeletonization or reconstruction of the full geometry of the entire network is required. The method was validated by using many isotropic in silico generated networks of different structures, morphologies, and concentrations. For each type of network, the method provides accurate estimates (a few percent) of the average and the standard deviation of the three-dimensional distribution of the pore sizes, defined as the diameters of the largest spheres that can be fit into the pore zones of the entire gel volume. When applied to the analysis of real confocal microscopy images taken on fibrin gels, the method provides an estimate of ξ0 consistent with results from elastic light scattering data.

  5. Comparisons of Gas-phase Temperature Measurements in a Flame Using Thin-Filament Pyrometry and Thermocouples

    NASA Technical Reports Server (NTRS)

    Struk, Peter; Dietrich, Daniel; Valentine, Russell; Feier, Ioan

    2003-01-01

    Less-intrusive, fast-responding, and full-field temperature measurements have long been a desired tool for the research community. Recently, the emission of a silicon-carbide (SiC) fiber placed in a flowing hot (or reacting) gas has been used to measure the temperature profile along the length of the fiber. The relationship between the gas and fiber temperature comes from an energy balance on the fiber. In the present work, we compared single point flame temperature measurements using thin-filament pyrometry (TFP) and thermocouples. The data was from vertically traversing a thermocouple and a SiC fiber through a methanol/air diffusion flame of a porous-metal wick burner. The results showed that the gas temperature using the TFP technique agreed with the thermocouple measurements (25.4 m diameter wire) within 3.5% for temperatures above 1200 K. Additionally, we imaged the entire SiC fiber (with a spatial resolution of 0.14 mm) while it was in the flame using a high resolution CCD camera. The intensity level along the fiber length is a function of the temperature. This results in a one-dimensional temperature profiles at various heights above the burner wick. This temperature measurement technique, while having a precision of less than 1 K, showed data scatter as high as 38 K. Finally, we discuss the major sources of uncertainty in gas temperature measurement using TFP.

  6. Actin network architecture can determine myosin motor activity.

    PubMed

    Reymann, Anne-Cécile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guérin, Christophe; Cao, Wenxiang; Chin, Harvey F; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2012-06-08

    The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

  7. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar); Skin lesion - actinic keratosis ... likely to develop it if you: Have fair skin, blue or green eyes, or blond or red ...

  8. Solid friction between soft filaments

    NASA Astrophysics Data System (ADS)

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A. W. C.; Vitelli, Vincenzo; Mahadevan, L.; Dogic, Zvonimir

    2015-06-01

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments’ overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes’s drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. Our findings demonstrate how altering a filament’s elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.

  9. Solid friction between soft filaments

    SciTech Connect

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A. W. C.; Vitelli, Vincenzo; Mahadevan, L.; Dogic, Zvonimir

    2015-03-02

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments’ overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes’s drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. In conclusion, our findings demonstrate how altering a filament’s elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.

  10. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

    Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

    2001-05-01

    Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

  11. Choice of tracks, microtubules and/or actin filaments for chloroplast photo-movement is differentially controlled by phytochrome and a blue light receptor.

    PubMed

    Sato, Y; Wada, M; Kadota, A

    2001-01-01

    Light induced chloroplast movement has been studied as a model system for photoreception and actin microfilament (MF)-based intracellular motilities in plants. Chloroplast photo-accumulation and -avoidance movement is mediated by phytochrome as well as blue light (BL) receptor in the moss Physcomitrella patens. Here we report the discovery of an involvement of a microtubule (MT)-based system in addition to an MF-based system in photorelocation of chloroplasts in this moss. In the dark, MTs provided tracks for rapid movement of chloroplasts in a longitudinal direction and MFs contributed the tracks for slow movement in any direction. We found that phytochrome responses utilized only the MT-based system, while BL responses had an alternative way of moving, either along MTs or MFs. MT-based systems were mediated by both photoreceptors, but chloroplasts showed movements with different velocity and pattern between them. No apparent difference in the behavior of chloroplast movement between the accumulation and avoidance movement was detected in phytochrome responses or BL responses, except for the direction of the movement. The results presented here demonstrate that chloroplasts use both MTs and MFs for motility and that phytochrome and a BL receptor control directional photo-movement of chloroplasts through the differential regulation of these motile systems.

  12. Branching of keratin intermediate filaments.

    PubMed

    Nafeey, Soufi; Martin, Ines; Felder, Tatiana; Walther, Paul; Felder, Edward

    2016-06-01

    Keratin intermediate filaments (IFs) are crucial to maintain mechanical stability in epithelial cells. Since little is known about the network architecture that provides this stiffness and especially about branching properties of filaments, we addressed this question with different electron microscopic (EM) methods. Using EM tomography of high pressure frozen keratinocytes, we investigated the course of several filaments in a branching of a filament bundle. Moreover we found several putative bifurcations in individual filaments. To verify our observation we also visualized the keratin network in detergent extracted keratinocytes with scanning EM. Here bifurcations of individual filaments could unambiguously be identified additionally to bundle branchings. Interestingly, identical filament bifurcations were also found in purified keratin 8/18 filaments expressed in Escherichia coli which were reassembled in vitro. This excludes that an accessory protein contributes to the branch formation. Measurements of the filament cross sectional areas showed various ratios between the three bifurcation arms. This demonstrates that intermediate filament furcation is very different from actin furcation where an entire new filament is attached to an existing filament. Instead, the architecture of intermediate filament bifurcations is less predetermined and hence consistent with the general concept of IF formation.

  13. Reversible stress softening of actin networks

    PubMed Central

    Chaudhuri, Ovijit; Parekh, Sapun H.; Fletcher, Daniel A.

    2011-01-01

    The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes1–3. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension4–8. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks. PMID:17230186

  14. Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans.

    PubMed

    Ketelaar, Tijs; Meijer, Harold J G; Spiekerman, Marjolein; Weide, Rob; Govers, Francine

    2012-12-01

    The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin

  15. Polymerization of actin by positively charged liposomes

    PubMed Central

    1988-01-01

    By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N- pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts. PMID:3360852

  16. Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

    PubMed Central

    Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

    2015-01-01

    Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

  17. Effects on shortening velocity of rabbit skeletal muscle due to variations in the level of thin-filament activation.

    PubMed Central

    Moss, R L

    1986-01-01

    The effect of Ca2+ upon maximum shortening velocity (Vmax) has been investigated in skinned single fibres from rabbit psoas muscles. Vmax was obtained at 15 degrees C by measuring the amounts of time (delta t) required to take up various amounts of slack (delta l) imposed at one end of the fibre. During maximal activation with Ca2+, plots of delta l vs. delta t were well fitted by a single straight line. Calculation of Vmax from the slopes of the fitted lines yielded a Vmax of 4.44 +/- 0.15 (S.E. of mean) muscle lengths per second (m.l./s). However, at lower levels of Ca2+ activation, plots of delta l vs. delta t were biphasic, containing an initial phase of steady high-velocity shortening and a subsequent phase of steady low-velocity shortening. The transition between these two phases occurred following active shortening equivalent to 60-80 nm/half-sarcomere. Vmax during the high-velocity phase was relatively insensitive to Ca2+ concentration between pCas (i.e. -log [Ca2+]) of 4.5 and 6.0; however, Vmax fell to 3.58 +/- 0.40 m.l./s at pCa 6.1 and further to 1.02 +/- 0.30 m.l./s at pCa 6.2. Vmax during the low-velocity phase decreased as Ca2+ was lowered within the entire range of pCas studied to a minimum value of 0.35 +/- 0.09 m.l./s at pCa 6.2. The degree of thin-filament activation at a particular pCa was varied by partial extraction of troponin-C, which resulted in a permanent though reversible inactivation of parts of the thin filaments. Partial extraction of troponin-C altered the plots of delta l vs. delta t obtained at pCa 4.5 to a biphasic form. In addition, Vmax during the high- and low-velocity phases of shortening was reduced at each pCa greater than 4.5. Vmax values obtained in control fibres at low Ca2+ concentrations and extracted fibres were in good agreement when generated isometric tensions were equivalent. This was the case for both the high- and low-velocity phases of shortening. Fibres were also activated in the absence of Ca2+ by partial

  18. The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

    PubMed Central

    Belmont, Lisa D.; Drubin, David G.

    1998-01-01

    Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables. PMID:9732289

  19. Cytoplasmic Actin: Purification and Single Molecule Assembly Assays

    PubMed Central

    Hansen, Scott D.; Zuchero, J. Bradley; Mullins, R. Dyche

    2014-01-01

    The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy. PMID:23868587

  20. Actin Capping Protein is required for Dendritic Spine Development and Synapse Formation

    PubMed Central

    Fan, Yanjie; Tang, Xin; Vitriol, Eric; Chen, Gong; Zheng, James Q.

    2011-01-01

    Dendritic spines serve as the postsynaptic platform for most excitatory synapses in the mammalian brain and their shape and size are tightly correlated with synaptic strength. The actin cytoskeleton plays a crucial role in the spine structure and its modifications during synapse development and plasticity, but the underlying regulatory mechanisms remain to be elucidated. Here we report that actin capping protein (CP), a regulator of actin filament growth, plays an essential role for spine development and synapse formation. We found that CP expression in rat hippocampus is elevated at and after the stage of substantial synapse formation. CP knockdown in hippocampal cultures resulted in a marked decline in the spine density accompanied by increased filopodia-like protrusions. Moreover, the spines of CP knockdown neurons exhibited an altered morphology, highlighted by multiple thin filopodia-like protrusions emerging from the spine head. Finally, the number of functional synapses was reduced by CP knockdown as evidenced by a reduction in the density of paired pre- and postsynaptic markers and in the frequency of miniature excitatory postsynaptic currents. These findings indicate that capping of actin filaments by CP represents an essential step for the remodeling of the actin architecture underlying spine morphogenesis and synaptic formation during development. PMID:21752999

  1. Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis.

    PubMed

    Kadota, Akeo; Yamada, Noboru; Suetsugu, Noriyuki; Hirose, Mana; Saito, Chieko; Shoda, Keiko; Ichikawa, Satoshi; Kagawa, Takatoshi; Nakano, Akihiko; Wada, Masamitsu

    2009-08-04

    Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement.

  2. Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis

    PubMed Central

    Kadota, Akeo; Yamada, Noboru; Suetsugu, Noriyuki; Hirose, Mana; Saito, Chieko; Shoda, Keiko; Ichikawa, Satoshi; Kagawa, Takatoshi; Nakano, Akihiko; Wada, Masamitsu

    2009-01-01

    Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement. PMID:19620714

  3. The Effects of Disease Models of Nuclear Actin Polymerization on the Nucleus

    PubMed Central

    Serebryannyy, Leonid A.; Yuen, Michaela; Parilla, Megan; Cooper, Sandra T.; de Lanerolle, Primal

    2016-01-01

    Actin plays a crucial role in regulating multiple processes within the nucleus, including transcription and chromatin organization. However, the polymerization state of nuclear actin remains controversial, and there is no evidence for persistent actin filaments in a normal interphase nucleus. Further, several disease pathologies are characterized by polymerization of nuclear actin into stable filaments or rods. These include filaments that stain with phalloidin, resulting from point mutations in skeletal α-actin, detected in the human skeletal disease intranuclear rod myopathy, and cofilin/actin rods that form in response to cellular stressors like heatshock. To further elucidate the effects of these pathological actin structures, we examined the nucleus in both cell culture models as well as isolated human tissues. We find these actin structures alter the distribution of both RNA polymerase II and chromatin. Our data suggest that nuclear actin filaments result in disruption of nuclear organization, which may contribute to the disease pathology. PMID:27774069

  4. Regulation of myosin II activity by actin architecture

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Stam, Samantha; McCall, Patrick; Munro, Edwin; Gardel, Margaret

    2015-03-01

    Networks of actin filaments containing myosin II motors generate forces and motions that promote biological processes such as cell division, motility, and cargo transport. In cells, actin filaments are arranged in various structures from disordered meshworks to tight bundles. Clusters of myosin II motors, known as myosin filaments, crosslink an